Science.gov

Sample records for 4-phosphate 5-kinase pip5k

  1. Nuclear pool of phosphatidylinositol 4 phosphate 5 kinase 1α is modified by polySUMO-2 during apoptosis

    SciTech Connect

    Chakrabarti, Rajarshi; Bhowmick, Debajit; Bhargava, Varsha; Bhar, Kaushik; Siddhanta, Anirban

    2013-09-20

    Highlights: •Nuclear pool of PIP5K is SUMOylated. •Enhancement of SUMOylated nuclear PIP5K during apoptosis. •Nuclear PIP5K is modified by polySUMO-1 during apoptosis. •Nuclear PIP5K is modified by polySUMO-2 chain during apoptosis. -- Abstract: Phosphatidylinositol 4 phosphate 5 kinase 1α (PIP5K) is mainly localized in the cytosol and plasma membrane. Studies have also indicated its prominent association with nuclear speckles. The exact nature of this nuclear pool of PIP5K is not clear. Using biochemical and microscopic techniques, we have demonstrated that the nuclear pool of PIP5K is modified by SUMO-1 in HEK-293 cells stably expressing PIP5K. Moreover, this SUMOylated pool of PIP5K increased during apoptosis. PolySUMO-2 chain conjugated PIP5K was detected by pull-down experiment using affinity-tagged RNF4, a polySUMO-2 binding protein, during late apoptosis.

  2. The lipid kinase PIP5K1C regulates pain signaling and sensitization

    PubMed Central

    Wright, Brittany D.; Loo, Lipin; Street, Sarah E.; Ma, Anqi; Taylor-Blake, Bonnie; Stashko, Michael A.; Jin, Jian; Janzen, William P.; Frye, Stephen V.; Zylka, Mark J.

    2014-01-01

    SUMMARY Numerous pain-producing (pronociceptive) receptors signal via phosphatidylinositol 4,5- bisphosphate (PIP2) hydrolysis. However, it is currently unknown which lipid kinases generate PIP2 in nociceptive dorsal root ganglia (DRG) neurons and if these kinases regulate pronociceptive receptor signaling. Here, we found that phosphatidylinositol 4-phosphate 5 kinase type 1C (PIP5K1C) is expressed at higher levels than any other PIP5K and, based on experiments with Pip5k1c+/− mice, generates at least half of all PIP2 in DRG neurons. Additionally, Pip5k1c haploinsufficiency reduces pronociceptive receptor signaling and TRPV1 sensitization in DRG neurons as well as thermal and mechanical hypersensitivity in mouse models of chronic pain. We identified a novel small molecule inhibitor of PIP5K1C (UNC3230) in a high-throughput screen. UNC3230 lowered PIP2 levels in DRG neurons and attenuated hypersensitivity when administered intrathecally or into the hindpaw. Our studies reveal that PIP5K1C regulates PIP2- dependent nociceptive signaling and suggest that PIP5K1C is a novel therapeutic target for chronic pain. PMID:24853942

  3. Development of a High-Throughput Screening Assay to Identify Inhibitors of the Lipid Kinase PIP5K1C.

    PubMed

    Wright, Brittany D; Simpson, Catherine; Stashko, Michael; Kireev, Dmitri; Hull-Ryde, Emily A; Zylka, Mark J; Janzen, William P

    2015-06-01

    Phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) regulate a variety of cellular processes, including signaling through G protein-coupled receptors (GPCRs), endocytosis, exocytosis, and cell migration. These lipid kinases synthesize phosphatidylinositol 4,5-bisphosphate (PIP2) from phosphatidylinositol 4-phosphate [PI(4)P]. Because small-molecule inhibitors of these lipid kinases did not exist, molecular and genetic approaches were predominantly used to study PIP5K1 regulation of these cellular processes. Moreover, standard radioisotope-based lipid kinase assays cannot be easily adapted for high-throughput screening. Here, we report a novel, high-throughput, microfluidic mobility shift assay to identify inhibitors of PIP5K1C. This assay uses fluorescently labeled phosphatidylinositol 4-phosphate as the substrate and recombinant human PIP5K1C. Our assay exhibited high reproducibility, had a calculated adenosine triphosphate Michaelis constant (Km) of 15 µM, performed with z' values >0.7, and was used to screen a kinase-focused library of ~4700 compounds. From this screen, we identified several potent inhibitors of PIP5K1C, including UNC3230, a compound that we recently found can reduce nociceptive sensitization in animal models of chronic pain. This novel assay will allow continued drug discovery efforts for PIP5K1C and can be adapted easily to screen additional lipid kinases.

  4. Development of a high-throughput screening assay to identify inhibitors of the lipid kinase PIP5K1C

    PubMed Central

    Wright, Brittany D.; Simpson, Catherine; Stashko, Michael; Kireev, Dmitri; Hull-Ryde, Emily A.; Zylka, Mark J.; Janzen, William P.

    2015-01-01

    Phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) regulate a variety of cellular processes including signaling through G protein-coupled receptors (GPCRs), endocytosis, exocytosis, and cell migration. These lipid kinases synthesize phosphatidylinositol 4,5-bisphosphate (PIP2) from phosphatidylinositol 4-phosphate [PI(4)P]. Since small molecule inhibitors of these lipid kinases did not exist, molecular and genetic approaches were predominantly used to study PIP5K1 regulation of these cellular processes. Moreover, standard radioisotope-based lipid kinase assays cannot be easily adapted for high-throughput screening. Here, we report a novel high-throughput microfluidic mobility shift assay to identify inhibitors of PIP5K1C. This assay utilizes fluorescently labeled phosphatidylinositol 4-phosphate as the substrate and recombinant human PIP5K1C. Our assay exhibited high reproducibility, had a calculated ATP Km of 15 µM, performed with z’ values >0.7, and was used to screen a kinase-focused library of ~4,700 compounds. From this screen, we identified several potent inhibitors of PIP5K1C, including UNC3230, a compound that we recently found can reduce nociceptive sensitization in animal models of chronic pain. This novel assay will allow continued drug discovery efforts for PIP5K1C and can be easily adapted to screen additional lipid kinases. PMID:25534829

  5. The activation loop of PIP5K functions as a membrane sensor essential for lipid substrate processing

    PubMed Central

    Liu, Aizhuo; Sui, Dexin; Wu, Dianqing; Hu, Jian

    2016-01-01

    Phosphatidylinositol 4-phosphate 5-kinase (PIP5K), a representative member of the phosphatidylinositol phosphate kinase (PIPK) family, is a major enzyme that biosynthesizes the signaling molecule PI(4,5)P2 (phosphatidylinositol 4,5-bisphosphate) in eukaryotic cells. The stringent specificity toward lipid substrates and the high sensitivity to the membrane environment strongly suggest a membrane-sensing mechanism, but the underlying structural basis is still largely unknown. We present a nuclear magnetic resonance (NMR) study on a peptide commensurate with a PIP5K’s activation loop, which has been reported to be a determinant of lipid substrate specificity and subcellular localization of PIP5K. Although the activation loop is severely disordered in the crystal structure of PIP5K, the NMR experiments showed that the largely unstructured peptide folded into an amphipathic helix upon its association with the 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) micellar surface. Systematic mutagenesis and functional assays further demonstrated the crucial roles of the amphipathic helix and its hydrophobic surface in kinase activity and membrane-sensing function, supporting a working model in which the activation loop is a critical structural module conferring a membrane-sensing mechanism on PIP5K. The activation loop, surprisingly functioning as a membrane sensor, represents a new paradigm of kinase regulation by the activation loop through protein-membrane interaction, which also lays a foundation on the regulation of PIP5K (and other PIPKs) by membrane lipids for future studies. PMID:28138522

  6. The role of PI3K/AKT-related PIP5K1α and the discovery of its selective inhibitor for treatment of advanced prostate cancer.

    PubMed

    Semenas, Julius; Hedblom, Andreas; Miftakhova, Regina R; Sarwar, Martuza; Larsson, Rikard; Shcherbina, Liliya; Johansson, Martin E; Härkönen, Pirkko; Sterner, Olov; Persson, Jenny L

    2014-09-02

    Nitrogen-containing heterocyclic compounds are an important class of molecules that are commonly used for the synthesis of candidate drugs. Phosphatidylinositol-4-phosphate 5-kinase-α (PIP5Kα) is a lipid kinase, similar to PI3K. However, the role of PIP5K1α in oncogenic processes and the development of inhibitors that selectively target PIP5K1α have not been reported. In the present study we report that overexpression of PIP5K1α is associated with poor prognosis in prostate cancer and correlates with an elevated level of the androgen receptor. Overexpression of PIP5K1α in PNT1A nonmalignant cells results in an increased AKT activity and an increased survival, as well as invasive malignant phenotype, whereas siRNA-mediated knockdown of PIP5K1α in aggressive PC-3 cells leads to a reduced AKT activity and an inhibition in tumor growth in xenograft mice. We further report a previously unidentified role for PIP5K1α as a druggable target for our newly developed compound ISA-2011B using a high-throughput KINOMEscan platform. ISA-2011B was discovered during our synthetic studies of C-1 indol-3-yl substituted 1,2,3,4-tetrahydroisoquinolines via a Pictet-Spengler approach. ISA-2011B significantly inhibits growth of tumor cells in xenograft mice, and we show that this is mediated by targeting PIP5K1α-associated PI3K/AKT and the downstream survival, proliferation, and invasion pathways. Further, siRNA-mediated knockdown of PIP5K1α exerts similar effects on PC3 cells as ISA-2011B treatment, significantly inhibiting AKT activity, increasing apoptosis and reducing invasion. Thus, PIP5K1α has high potential as a drug target, and compound ISA-2011B is interesting for further development of targeted cancer therapy.

  7. The role of PI3K/AKT-related PIP5K1α and the discovery of its selective inhibitor for treatment of advanced prostate cancer

    PubMed Central

    Semenas, Julius; Hedblom, Andreas; Miftakhova, Regina R.; Sarwar, Martuza; Larsson, Rikard; Shcherbina, Liliya; Johansson, Martin E.; Härkönen, Pirkko; Sterner, Olov; Persson, Jenny L.

    2014-01-01

    Nitrogen-containing heterocyclic compounds are an important class of molecules that are commonly used for the synthesis of candidate drugs. Phosphatidylinositol-4-phosphate 5-kinase-α (PIP5Kα) is a lipid kinase, similar to PI3K. However, the role of PIP5K1α in oncogenic processes and the development of inhibitors that selectively target PIP5K1α have not been reported. In the present study we report that overexpression of PIP5K1α is associated with poor prognosis in prostate cancer and correlates with an elevated level of the androgen receptor. Overexpression of PIP5K1α in PNT1A nonmalignant cells results in an increased AKT activity and an increased survival, as well as invasive malignant phenotype, whereas siRNA-mediated knockdown of PIP5K1α in aggressive PC-3 cells leads to a reduced AKT activity and an inhibition in tumor growth in xenograft mice. We further report a previously unidentified role for PIP5K1α as a druggable target for our newly developed compound ISA-2011B using a high-throughput KINOMEscan platform. ISA-2011B was discovered during our synthetic studies of C-1 indol-3-yl substituted 1,2,3,4-tetrahydroisoquinolines via a Pictet-Spengler approach. ISA-2011B significantly inhibits growth of tumor cells in xenograft mice, and we show that this is mediated by targeting PIP5K1α-associated PI3K/AKT and the downstream survival, proliferation, and invasion pathways. Further, siRNA-mediated knockdown of PIP5K1α exerts similar effects on PC3 cells as ISA-2011B treatment, significantly inhibiting AKT activity, increasing apoptosis and reducing invasion. Thus, PIP5K1α has high potential as a drug target, and compound ISA-2011B is interesting for further development of targeted cancer therapy. PMID:25071204

  8. Type I phosphatidylinositol 4-phosphate 5-kinase homo- and heterodimerization determines its membrane localization and activity.

    PubMed

    Lacalle, Rosa Ana; de Karam, Juan C; Martínez-Muñoz, Laura; Artetxe, Ibai; Peregil, Rosa M; Sot, Jesús; Rojas, Ana M; Goñi, Félix M; Mellado, Mario; Mañes, Santos

    2015-06-01

    Type I phosphatidylinositol 4-phosphate 5-kinases (PIP5KIs; α, β, and γ) are a family of isoenzymes that produce phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] using phosphatidylinositol 4-phosphate as substrate. Their structural homology with the class II lipid kinases [type II phosphatidylinositol 5-phosphate 4-kinase (PIP4KII)] suggests that PIP5KI dimerizes, although this has not been formally demonstrated. Neither the hypothetical structural dimerization determinants nor the functional consequences of dimerization have been studied. Here, we used Förster resonance energy transfer, coprecipitation, and ELISA to show that PIP5KIβ forms homo- and heterodimers with PIP5KIγ_i2 in vitro and in live human cells. Dimerization appears to be a general phenomenon for PIP5KI isoenzymes because PIP5KIβ/PIP5KIα heterodimers were also detected by mass spectrometry. Dimerization was independent of actin cytoskeleton remodeling and was also observed using purified proteins. Mutagenesis studies of PIP5KIβ located the dimerization motif at the N terminus, in a region homologous to that implicated in PIP4KII dimerization. PIP5KIβ mutants whose dimerization was impaired showed a severe decrease in PI(4,5)P2 production and plasma membrane delocalization, although their association to lipid monolayers was unaltered. Our results identify dimerization as an integral feature of PIP5K proteins and a central determinant of their enzyme activity.

  9. Targeted suppression of AR-V7 using PIP5K1α inhibitor overcomes enzalutamide resistance in prostate cancer cells

    PubMed Central

    Sarwar, Martuza; Semenas, Julius; Miftakhova, Regina; Simoulis, Athanasios; Robinson, Brian; Wingren, Anette Gjörloff; Mongan, Nigel P.; Heery, David M.; Johnsson, Heather; Abrahamsson, Per-Anders; Dizeyi, Nishtman; Luo, Jun; Persson, Jenny L.

    2016-01-01

    One mechanism of resistance of prostate cancer (PCa) to enzalutamide (MDV3100) treatment is the increased expression of AR variants lacking the ligand binding-domain, the best characterized of which is AR-V7. We have previously reported that Phosphatidylinositol-4-phosphate 5-kinase alpha (PIP5Kα), is a lipid kinase that links to CDK1 and AR pathways. The discovery of PIP5Kα inhibitor highlight the potential of PIP5K1α as a drug target in PCa. In this study, we show that AR-V7 expression positively correlates with PIP5K1α in tumor specimens from PCa patients. Overexpression of AR-V7 increases PIP5K1α, promotes rapid growth of PCa in xenograft mice, whereas inhibition of PIP5K1α by its inhibitor ISA-2011B suppresses the growth and invasiveness of xenograft tumors overexpressing AR-V7. PIP5K1α is a key co-factor for both AR-V7 and AR, which are present as protein-protein complexes predominantly in the nucleus of PCa cells. In addition, PIP5K1α and CDK1 influence AR-V7 expression also through AKT-associated mechanism dependent on PTEN-status. ISA-2011B disrupts protein stabilization of AR-V7 which is dependent on PIP5K1α, leading to suppression of invasive growth of AR-V7-high tumors in xenograft mice. Our study suggests that combination of enzalutamide and PIP5K1α may have a significant impact on refining therapeutic strategies to circumvent resistance to antiandrogen therapies. PMID:27588408

  10. Targeted suppression of AR-V7 using PIP5K1α inhibitor overcomes enzalutamide resistance in prostate cancer cells.

    PubMed

    Sarwar, Martuza; Semenas, Julius; Miftakhova, Regina; Simoulis, Athanasios; Robinson, Brian; Gjörloff Wingren, Anette; Mongan, Nigel P; Heery, David M; Johnsson, Heather; Abrahamsson, Per-Anders; Dizeyi, Nishtman; Luo, Jun; Persson, Jenny L

    2016-09-27

    One mechanism of resistance of prostate cancer (PCa) to enzalutamide (MDV3100) treatment is the increased expression of AR variants lacking the ligand binding-domain, the best characterized of which is AR-V7. We have previously reported that Phosphatidylinositol-4-phosphate 5-kinase alpha (PIP5Kα), is a lipid kinase that links to CDK1 and AR pathways. The discovery of PIP5Kα inhibitor highlight the potential of PIP5K1α as a drug target in PCa. In this study, we show that AR-V7 expression positively correlates with PIP5K1α in tumor specimens from PCa patients. Overexpression of AR-V7 increases PIP5K1α, promotes rapid growth of PCa in xenograft mice, whereas inhibition of PIP5K1α by its inhibitor ISA-2011B suppresses the growth and invasiveness of xenograft tumors overexpressing AR-V7. PIP5K1α is a key co-factor for both AR-V7 and AR, which are present as protein-protein complexes predominantly in the nucleus of PCa cells. In addition, PIP5K1α and CDK1 influence AR-V7 expression also through AKT-associated mechanism dependent on PTEN-status. ISA-2011B disrupts protein stabilization of AR-V7 which is dependent on PIP5K1α, leading to suppression of invasive growth of AR-V7-high tumors in xenograft mice. Our study suggests that combination of enzalutamide and PIP5K1α may have a significant impact on refining therapeutic strategies to circumvent resistance to antiandrogen therapies.

  11. Identification of a Conserved 8 aa Insert in the PIP5K Protein in the Saccharomycetaceae family of Fungi and the Molecular Dynamics Simulations and Structural Analysis to investigate its Potential Functional Role.

    PubMed

    Khadka, Bijendra; Gupta, Radhey S

    2017-04-13

    Homologs of the phosphatidylinositol-4-phosphate-5-kinase (PIP5K), which controls a multitude of essential cellular functions, contain a 8 aa insert in a conserved region that is specific for the Saccharomycetaceae family of fungi. Using structures of human PIP4K proteins as templates, structural models were generated of the Saccharomyces cerevisiae and human PIP5K proteins. In the modeled S. cerevisiae PIP5K, the 8 aa insert forms a surface exposed loop, present on the same face of the protein as the activation loop of the kinase domain. Electrostatic potential analysis indicates that the residues from 8 aa conserved loop form a highly-positively charged surface patch, which through electrostatic interaction with the anionic portions of phospholipid head groups, is expected to play a role in the membrane interaction of the yeast PIP5K. To unravel this prediction, molecular dynamics (MD) simulations were carried out to examine the binding interaction of PIP5K, either containing or lacking the conserved signature insert (CSI), with two different membrane lipid bilayers. The results from MD studies provide insights concerning the mechanistic of interaction of PIP5K with lipid bilayer, and support the contention that the identified 8 aa conserved insert in fungal PIP5K plays an important role in the binding of this protein with membrane surface. This article is protected by copyright. All rights reserved.

  12. ARC3, a chloroplast division factor, is a chimera of prokaryotic FtsZ and part of eukaryotic phosphatidylinositol-4-phosphate 5-kinase.

    PubMed

    Shimada, Hiroshi; Koizumi, Masato; Kuroki, Kouta; Mochizuki, Mariko; Fujimoto, Hitoshi; Ohta, Hiroyuki; Masuda, Tatsuru; Takamiya, Ken-ichiro

    2004-08-01

    The arc3 (accumulation and replication of chloroplast) mutant of Arabidopsis thaliana has a small number of abnormally large chloroplasts in the cell, suggesting that chloroplast division is arrested in the mutant and ARC3 has an important role in the initiation of chloroplast division. To elucidate the role of ARC3, first we identified the ARC3 gene, and determined the location of ARC3 protein during chloroplast division because the localization and spatial orientation of such division factors are vital for correct chloroplast division. Sequencing analysis showed that ARC3 was a fusion of the prokaryotic FtsZ and part of the eukaryotic phosphatidylinositol-4-phosphate 5-kinase (PIP5K) genes. The PIP5K-homologous region of ARC3 had no catalytic domain but a membrane-occupation-and-recognition-nexus (MORN) repeat motif. Immunofluorescence microscopy, Western blotting analysis and in vitro chloroplast import and protease protection assays revealed that ARC3 protein was soluble, and located on the outer surface of the chloroplast in a ring-like structure at the early stage of chloroplast division. Prokaryotes have one FtsZ as a gene for division but have no ARC3 counterparts, the chimera of FtsZ and PIP5K, suggesting that the ARC3 gene might have been generated from FtsZ as another division factor during the evolution of chloroplast by endosymbiosis.

  13. Phosphatidylinositol 4-phosphate 5-kinases 1 and 2 are involved in the regulation of vacuole morphology during Arabidopsis thaliana pollen development.

    PubMed

    Ugalde, José-Manuel; Rodriguez-Furlán, Cecilia; Rycke, Riet De; Norambuena, Lorena; Friml, Jiří; León, Gabriel; Tejos, Ricardo

    2016-09-01

    The pollen grains arise after meiosis of pollen mother cells within the anthers. A series of complex structural changes follows, generating mature pollen grains capable of performing the double fertilization of the female megasporophyte. Several signaling molecules, including hormones and lipids, have been involved in the regulation and appropriate control of pollen development. Phosphatidylinositol 4-phophate 5-kinases (PIP5K), which catalyze the biosynthesis of the phosphoinositide PtdIns(4,5)P2, are important for tip polar growth of root hairs and pollen tubes, embryo development, vegetative plant growth, and responses to the environment. Here, we report a role of PIP5Ks during microgametogenesis. PIP5K1 and PIP5K2 are expressed during early stages of pollen development and their transcriptional activity respond to auxin in pollen grains. Early male gametophytic lethality to certain grade was observed in both pip5k1(-/-) and pip5k2(-/-) single mutants. The number of pip5k mutant alleles is directly related to the frequency of aborted pollen grains suggesting the two genes are involved in the same function. Indeed PIP5K1 and PIP5K2 are functionally redundant since homozygous double mutants did not render viable pollen grains. The loss of function of PIP5K1 and PIP5K2results in defects in vacuole morphology in pollen at the later stages and epidermal root cells. Our results show that PIP5K1, PIP5K2 and phosphoinositide signaling are important cues for early developmental stages and vacuole formation during microgametogenesis.

  14. Resolution of structure of PIP5K1A reveals molecular mechanism for its regulation by dimerization and dishevelled

    PubMed Central

    Hu, Jian; Yuan, Qianying; Kang, Xue; Qin, Yuanbo; Li, Lin; Ha, Ya; Wu, Dianqing

    2015-01-01

    Type I phosphatidylinositol phosphate kinase (PIP5K1) phosphorylates the head group of phosphatidylinositol 4-phosphate (PtdIns4P) to generate PtdIns4,5P2, which plays important roles in a wide range of cellular functions including Wnt signalling. However, the lack of its structural information has hindered the understanding of its regulation. Here we report the crystal structure of the catalytic domain of zebrafish PIP5K1A at 3.3 Å resolution. This molecule forms a side-to-side dimer. Mutagenesis study of PIP5K1A reveals two adjacent interfaces for the dimerization and interaction with the DIX domain of the Wnt signalling molecule dishevelled. Although these interfaces are located distally to the catalytic/substrate-binding site, binding to these interfaces either through dimerization or the interaction with DIX stimulates PIP5K1 catalytic activity. DIX binding additionally enhances PIP5K1 substrate binding. Thus, this study elucidates regulatory mechanisms for this lipid kinase and provides a paradigm for the understanding of PIP5K1 regulation by their interacting molecules. PMID:26365782

  15. Effects of polyamines and calcium and sodium ions on smooth muscle cytoskeleton-associated phosphatidylinositol (4)-phosphate 5-kinase.

    PubMed

    Chen, H; Baron, C B; Griffiths, T; Greeley, P; Coburn, R F

    1998-10-01

    In many different cell types, including smooth muscle cells (Baron et al., 1989, Am. J. Physiol., 256: C375-383; Baron et al., J. Pharmacol. Exp. Ther. 266: 8-15), phosphatidylinositol (4)-phosphate 5-kinase plays a critical role in the regulation of membrane concentrations of phosphatidylinositol (4,5)-bisphosphate and formation of inositol (1,4,5)-trisphosphate. In unstimulated porcine trachealis smooth muscle, 70% of total cellular phosphatidylinositol (4)-phosphate 5-kinase activity was associated with cytoskeletal proteins and only trace activity was detectable in isolated sarcolemma. Using two different preparations, we studied cytoskeleton-associated phosphatidyl inositol (4)-phosphate 5-kinase under conditions that attempted to mimic the ionic and thermal cytoplasmic environment of living cells. The cytoskeleton-associated enzyme, studied using phosphatidylinositol (4)-phosphate substrate concentrations that produced phosphatidylinositol 4,5-bisphosphate at about 10% of the maximal rate, was sensitive to free [Mg2+], had an absolute requirement for phosphatidylserine, phosphatidic acid, or phosphatidylinositol, and included type I isoforms. At 0.5 mM free [Mg2+], physiological spermine concentrations, 0.2-0.4 mM, increased phosphatidylinositol (4)-phosphate 5-kinase activity two to four times compared to controls run without spermine. The EC50 for spermine-evoked increases in activity was 0.17 +/- 0.02 mM. Spermine-evoked enzyme activity was a function of both free [Mg2+] and substrate concentration. Cytoskeleton-associated phosphatidylinositol (4)-phosphate 5-kinase was inhibited by free [Ca2+] over a physiological range for cytoplasm--10(-8) to 10(-5) M, an effect independent of the presence of calmodulin. Na+ over the range 20 to 50 mM also inhibited this enzyme activated by 5 mM Mg2+ but had no effect on spermine-activated enzyme. Na+, Ca2+, and spermine appear to be physiological modulators of smooth muscle cytoskeleton-bound phosphatidylinositol (4

  16. Phosphatidylinositol 4-phosphate 5-kinase α is induced in ganglioside-stimulated brain astrocytes and contributes to inflammatory responses

    PubMed Central

    Kim, Bokyung; Yoon, Sarah; Kim, Yeon Joo; Liu, Tian; Woo, Joo Hong; Chwae, Yong-Joon; Joe, Eun-hye; Jou, Ilo

    2010-01-01

    In brain tissue, astrocytes play defensive roles in central nervous system integrity by mediating immune responses against pathological conditions. Type I phosphatidylinositol 4-phosphate 5-kinase α (PIP5Kα) that is responsible for production of phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) regulates many important cell functions at the cell surface. Here, we have examined whether PIP5Kα is associated with astrocyte inflammatory responses. Gangliosides are releasable from damaged cell membranes of neurons and capable of inducing inflammatory responses. We found that treatment of primary cultured astrocytes with gangliosides significantly enhanced PIP5Kα mRNA and protein expression levels. PI(4,5)P2 imaging using a fluorescent tubby (R332H) expression as a PI(4,5)P2-specific probe showed that ganglioside treatment increased PI(4,5)P2 level. Interestingly, microRNA-based PIP5Kα knockdown strongly reduced ganglioside-induced transcription of proinflammatory cytokines IL-1β and TNFα. PIP5Kα knockdown also suppressed ganglioside-induced phosphorylation and nuclear translocation of NF-κB and the degradation of IκB-α, indicating that PIP5Kα knockdown interfered with the ganglioside-activated NF-κB signaling. Together, these results suggest that PIP5Kα is a novel inflammatory mediator that undergoes upregulation and contributes to immune responses by facilitating NF-κB activation in ganglioside-stimulated astrocytes. PMID:20720456

  17. Association of protein kinase Cmu with type II phosphatidylinositol 4-kinase and type I phosphatidylinositol-4-phosphate 5-kinase.

    PubMed

    Nishikawa, K; Toker, A; Wong, K; Marignani, P A; Johannes, F J; Cantley, L C

    1998-09-04

    Protein kinase Cmu (PKCmu), also named protein kinase D, is an unusual member of the PKC family that has a putative transmembrane domain and pleckstrin homology domain. This enzyme has a substrate specificity distinct from other PKC isoforms (Nishikawa, K., Toker, A., Johannes, F. J., Songyang, Z., and Cantley, L. C. (1997) J. Biol. Chem. 272, 952-960), and its mechanism of regulation is not yet clear. Here we show that PKCmu forms a complex in vivo with a phosphatidylinositol 4-kinase and a phosphatidylinositol-4-phosphate 5-kinase. A region of PKCmu between the amino-terminal transmembrane domain and the pleckstrin homology domain is shown to be involved in the association with the lipid kinases. Interestingly, a kinase-dead point mutant of PKCmu failed to associate with either lipid kinase activity, indicating that autophosphorylation may be required to expose the lipid kinase interaction domain. Furthermore, the subcellular distribution of the PKCmu-associated lipid kinases to the particulate fraction depends on the presence of the amino-terminal region of PKCmu including the predicted transmembrane region. These results suggest a novel model in which the non-catalytic region of PKCmu acts as a scaffold for assembly of enzymes involved in phosphoinositide synthesis at specific membrane locations.

  18. Phosphatidylinositol phosphate 5-kinase genes respond to phosphate deficiency for root hair elongation in Arabidopsis thaliana.

    PubMed

    Wada, Yukika; Kusano, Hiroaki; Tsuge, Tomohiko; Aoyama, Takashi

    2015-02-01

    Plants drastically alter their root system architecture to adapt to different underground growth conditions. During phosphate (Pi) deficiency, most plants including Arabidopsis thaliana enhance the development of lateral roots and root hairs, resulting in bushy and hairy roots. To elucidate the signal pathway specific for the root hair elongation response to Pi deficiency, we investigated the expression of type-B phosphatidylinositol phosphate 5-kinase (PIP5K) genes, as a quantitative factor for root hair elongation in Arabidopsis. At young seedling stages, the PIP5K3 and PIP5K4 genes responded to Pi deficiency in steady-state transcript levels via PHR1-binding sequences (P1BSs) in their upstream regions. Both pip5k3 and pip5k4 single mutants, which exhibit short-root-hair phenotypes, remained responsive to Pi deficiency for root hair elongation; however the pip5k3pip5k4 double mutant exhibited shorter root hairs than the single mutants, and lost responsiveness to Pi deficiency at young seedling stages. In the tactical complementation line in which modified PIP5K3 and PIP5K4 genes with base substitutions in their P1BSs were co-introduced into the double mutant, root hairs of young seedlings had normal lengths under Pi-sufficient conditions, but were not responsive to Pi deficiency. From these results, we conclude that a Pi-deficiency signal is transferred to the pathway for root hair elongation via the PIP5K genes.

  19. Protein Kinase D1 regulates focal adhesion dynamics and cell adhesion through Phosphatidylinositol-4-phosphate 5-kinase type-l γ

    PubMed Central

    Durand, Nisha; Bastea, Ligia I.; Long, Jason; Döppler, Heike; Ling, Kun; Storz, Peter

    2016-01-01

    Focal adhesions (FAs) are highly dynamic structures that are assembled and disassembled on a continuous basis. The balance between the two processes mediates various aspects of cell behavior, ranging from cell adhesion and spreading to directed cell migration. The turnover of FAs is regulated at multiple levels and involves a variety of signaling molecules and adaptor proteins. In the present study, we show that in response to integrin engagement, a subcellular pool of Protein Kinase D1 (PKD1) localizes to the FAs. PKD1 affects FAs by decreasing turnover and promoting maturation, resulting in enhanced cell adhesion. The effects of PKD1 are mediated through direct phosphorylation of FA-localized phosphatidylinositol-4-phosphate 5-kinase type-l γ (PIP5Klγ) at serine residue 448. This phosphorylation occurs in response to Fibronectin-RhoA signaling and leads to a decrease in PIP5Klγs’ lipid kinase activity and binding affinity for Talin. Our data reveal a novel function for PKD1 as a regulator of FA dynamics and by identifying PIP5Klγ as a novel PKD1 substrate provide mechanistic insight into this process. PMID:27775029

  20. Protein Kinase D1 regulates focal adhesion dynamics and cell adhesion through Phosphatidylinositol-4-phosphate 5-kinase type-l γ.

    PubMed

    Durand, Nisha; Bastea, Ligia I; Long, Jason; Döppler, Heike; Ling, Kun; Storz, Peter

    2016-10-24

    Focal adhesions (FAs) are highly dynamic structures that are assembled and disassembled on a continuous basis. The balance between the two processes mediates various aspects of cell behavior, ranging from cell adhesion and spreading to directed cell migration. The turnover of FAs is regulated at multiple levels and involves a variety of signaling molecules and adaptor proteins. In the present study, we show that in response to integrin engagement, a subcellular pool of Protein Kinase D1 (PKD1) localizes to the FAs. PKD1 affects FAs by decreasing turnover and promoting maturation, resulting in enhanced cell adhesion. The effects of PKD1 are mediated through direct phosphorylation of FA-localized phosphatidylinositol-4-phosphate 5-kinase type-l γ (PIP5Klγ) at serine residue 448. This phosphorylation occurs in response to Fibronectin-RhoA signaling and leads to a decrease in PIP5Klγs' lipid kinase activity and binding affinity for Talin. Our data reveal a novel function for PKD1 as a regulator of FA dynamics and by identifying PIP5Klγ as a novel PKD1 substrate provide mechanistic insight into this process.

  1. The direct interaction between ASH2, a Drosophila trithorax group protein, and SKTL, a nuclear phosphatidylinositol 4-phosphate 5-kinase, implies a role for phosphatidylinositol 4,5-bisphosphate in maintaining transcriptionally active chromatin.

    PubMed Central

    Cheng, Mimi K; Shearn, Allen

    2004-01-01

    The products of trithorax group (trxG) genes maintain active transcription of many important developmental regulatory genes, including homeotic genes. Several trxG proteins have been shown to act in multimeric protein complexes that modify chromatin structure. ASH2, the product of the Drosophila trxG gene absent, small, or homeotic discs 2 (ash2) is a component of a 500-kD complex. In this article, we provide biochemical evidence that ASH2 binds directly to Skittles (SKTL), a predicted phosphatidylinositol 4-phosphate 5-kinase, and genetic evidence that the association of these proteins is functionally significant. We also show that histone H1 hyperphosphorylation is dramatically increased in both ash2 and sktl mutant polytene chromosomes. These results suggest that ASH2 maintains active transcription by binding a producer of nuclear phosphoinositides and downregulating histone H1 hyperphosphorylation. PMID:15280236

  2. Novel PI(4)P 5-kinase homologue, Fab1p, essential for normal vacuole function and morphology in yeast.

    PubMed

    Yamamoto, A; DeWald, D B; Boronenkov, I V; Anderson, R A; Emr, S D; Koshland, D

    1995-05-01

    The FAB1 gene of budding yeast is predicted to encode a protein of 257 kDa that exhibits significant sequence homology to a human type II PI(4)P 5-kinase (PIP5K-II). The recently cloned human PIP5K-II specifically converts PI(4)P to PI(4,5)P2 (Boronenkov and Anderson, 1995). The region of highest similarity between Fab1p and PIP5K-II includes a predicted nucleotide binding motif, which is likely to correspond to the catalytic domain of the protein. Interestingly, neither PIP5K-II nor Fab1p exhibit significant homology with cloned PI 3-kinases or PI 4-kinases. fab1 mutations result in the formation of aploid and binucleate cells (hence the name FAB). In addition, loss of Fab1p function causes defects in vacuole function and morphology, cell surface integrity, and cell growth. Experiments with a temperature conditional fab1 mutant revealed that their vacuoles rapidly (within 30 min) enlarge to more than double the size upon shifting cells to the nonpermissive temperature. Additional experiments with the fab1 ts mutant together with results obtained with fab1 vps (vacuolar protein sorting defective) double mutants indicate that the nuclear division and cell surface integrity defects observed in fab1 mutants are secondary to the vacuole morphology defects. Based on these data, we propose that Fab1p is a PI(4)P 5-kinase and that the product of the Fab1p reaction, PIP2, functions as an important regulator of vacuole homeostasis perhaps by controlling membrane flux to and/or from the vacuole. Furthermore, a comparison of the phenotypes of fab1 mutants and other yeast mutants affecting PI metabolism suggests that phosphoinositides may serve as general regulators of several different membrane trafficking pathways.

  3. Phosphatidylinositol 4,5-Bisphosphate Influences PIN Polarization by Controlling Clathrin-Mediated Membrane Trafficking in Arabidopsis[C][W

    PubMed Central

    Ischebeck, Till; Werner, Stephanie; Krishnamoorthy, Praveen; Lerche, Jennifer; Meijón, Mónica; Stenzel, Irene; Löfke, Christian; Wiessner, Theresa; Im, Yang Ju; Perera, Imara Y.; Iven, Tim; Feussner, Ivo; Busch, Wolfgang; Boss, Wendy F.; Teichmann, Thomas; Hause, Bettina; Persson, Staffan; Heilmann, Ingo

    2013-01-01

    The functions of the minor phospholipid phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] during vegetative plant growth remain obscure. Here, we targeted two related phosphatidylinositol 4-phosphate 5-kinases (PI4P 5-kinases) PIP5K1 and PIP5K2, which are expressed ubiquitously in Arabidopsis thaliana. A pip5k1 pip5k2 double mutant with reduced PtdIns(4,5)P2 levels showed dwarf stature and phenotypes suggesting defects in auxin distribution. The roots of the pip5k1 pip5k2 double mutant had normal auxin levels but reduced auxin transport and altered distribution. Fluorescence-tagged auxin efflux carriers PIN-FORMED (PIN1)–green fluorescent protein (GFP) and PIN2-GFP displayed abnormal, partially apolar distribution. Furthermore, fewer brefeldin A–induced endosomal bodies decorated by PIN1-GFP or PIN2-GFP formed in pip5k1 pip5k2 mutants. Inducible overexpressor lines for PIP5K1 or PIP5K2 also exhibited phenotypes indicating misregulation of auxin-dependent processes, and immunolocalization showed reduced membrane association of PIN1 and PIN2. PIN cycling and polarization require clathrin-mediated endocytosis and labeled clathrin light chain also displayed altered localization patterns in the pip5k1 pip5k2 double mutant, consistent with a role for PtdIns(4,5)P2 in the regulation of clathrin-mediated endocytosis. Further biochemical tests on subcellular fractions enriched for clathrin-coated vesicles (CCVs) indicated that pip5k1 and pip5k2 mutants have reduced CCV-associated PI4P 5-kinase activity. Together, the data indicate an important role for PtdIns(4,5)P2 in the control of clathrin dynamics and in auxin distribution in Arabidopsis. PMID:24326589

  4. A novel IRS-1-associated protein, DGKζ regulates GLUT4 translocation in 3T3-L1 adipocytes.

    PubMed

    Liu, TingYu; Yu, BuChin; Kakino, Mamoru; Fujimoto, Hitoshi; Ando, Yasutoshi; Hakuno, Fumihiko; Takahashi, Shin-Ichiro

    2016-10-14

    Insulin receptor substrates (IRSs) are major targets of insulin receptor tyrosine kinases. Here we identified diacylglycerol kinase zeta (DGKζ) as an IRS-1-associated protein, and examined roles of DGKζ in glucose transporter 4 (GLUT4) translocation to the plasma membrane. When DGKζ was knocked-down in 3T3-L1 adipocytes, insulin-induced GLUT4 translocation was inhibited without affecting other mediators of insulin-dependent signaling. Similarly, knockdown of phosphatidylinositol 4-phosphate 5-kinase 1α (PIP5K1α), which had been reported to interact with DGKζ, also inhibited insulin-induced GLUT4 translocation. Moreover, DGKζ interacted with IRS-1 without insulin stimulation, but insulin stimulation decreased this interaction. Over-expression of sDGKζ (short-form DGKζ), which competed out DGKζ from IRS-1, enhanced GLUT4 translocation without insulin stimulation. Taking these results together with the data showing that cellular PIP5K activity was correlated with GLUT4 translocation ability, we concluded that IRS-1-associated DGKζ prevents GLUT4 translocation in the absence of insulin and that the DGKζ dissociated from IRS-1 by insulin stimulation enhances GLUT4 translocation through PIP5K1α activity.

  5. A novel IRS-1-associated protein, DGKζ regulates GLUT4 translocation in 3T3-L1 adipocytes

    PubMed Central

    Liu, TingYu; Yu, BuChin; Kakino, Mamoru; Fujimoto, Hitoshi; Ando, Yasutoshi; Hakuno, Fumihiko; Takahashi, Shin-Ichiro

    2016-01-01

    Insulin receptor substrates (IRSs) are major targets of insulin receptor tyrosine kinases. Here we identified diacylglycerol kinase zeta (DGKζ) as an IRS-1-associated protein, and examined roles of DGKζ in glucose transporter 4 (GLUT4) translocation to the plasma membrane. When DGKζ was knocked-down in 3T3-L1 adipocytes, insulin-induced GLUT4 translocation was inhibited without affecting other mediators of insulin-dependent signaling. Similarly, knockdown of phosphatidylinositol 4-phosphate 5-kinase 1α (PIP5K1α), which had been reported to interact with DGKζ, also inhibited insulin-induced GLUT4 translocation. Moreover, DGKζ interacted with IRS-1 without insulin stimulation, but insulin stimulation decreased this interaction. Over-expression of sDGKζ (short-form DGKζ), which competed out DGKζ from IRS-1, enhanced GLUT4 translocation without insulin stimulation. Taking these results together with the data showing that cellular PIP5K activity was correlated with GLUT4 translocation ability, we concluded that IRS-1-associated DGKζ prevents GLUT4 translocation in the absence of insulin and that the DGKζ dissociated from IRS-1 by insulin stimulation enhances GLUT4 translocation through PIP5K1α activity. PMID:27739494

  6. Combination of vascular targeting PDT with combretastatin A4 phosphate

    NASA Astrophysics Data System (ADS)

    He, Chong; Fateye, Babasola; Chen, Bin

    2009-06-01

    Tumor vasculature is an attractive target for cancer therapy due to its accessibility to blood-borne therapeutic agents and the dependence of tumor cells on a functional blood supply for survival and growth. Vascular targeting photodynamic therapy (vPDT) is a novel modality based on the selective laser light activation of photosensitizers localized inside tumor vasculature to shutdown tumor vascular function. Although this vascular targeting therapy is showing great promise for cancer treatment, tumor recurrence has been observed in both preclinical and clinical studies. In this study, we intend to enhance the therapeutic outcome of vascular targeting PDT by combining it with combretastatin A4 phosphate (CA4P), a blood flow inhibitor. We found that the combination of CA4P and vPDT significantly increased endothelial cell apoptosis than each single therapy. Western blot analysis suggests that myosin light chain kinase (MLCK) is a common target of CA4P and vPDT. In a PC-3 prostate tumor model, we found that CA4P was able to greatly enhance tumor response to vPDT. These results demonstrate that CA4P and vPDT can be combined to enhance the therapeutic effect.

  7. Multiphasic dynamics of phosphatidylinositol 4-phosphate during phagocytosis

    PubMed Central

    Levin, Roni; Hammond, Gerald R. V.; Balla, Tamas; De Camilli, Pietro; Fairn, Gregory D.; Grinstein, Sergio

    2017-01-01

    We analyzed the distribution, fate, and functional role of phosphatidylinositol 4-phosphate (PtdIns4P) during phagosome formation and maturation. To this end, we used genetically encoded probes consisting of the PtdIns4P-binding domain of the bacterial effector SidM. PtdIns4P was found to undergo complex, multiphasic changes during phagocytosis. The phosphoinositide, which is present in the plasmalemma before engagement of the target particle, is transiently enriched in the phagosomal cup. Soon after the phagosome seals, PtdIns4P levels drop precipitously due to the hydrolytic activity of Sac2 and phospholipase C, becoming undetectable for ∼10 min. PtdIns4P disappearance coincides with the emergence of phagosomal PtdIns3P. Conversely, the disappearance of PtdIns3P that signals the transition from early to late phagosomes is accompanied by resurgence of PtdIns4P, which is associated with the recruitment of phosphatidylinositol 4-kinase 2A. The reacquisition of PtdIns4P can be prevented by silencing expression of the kinase and can be counteracted by recruitment of a 4-phosphatase with a heterodimerization system. Using these approaches, we found that the secondary accumulation of PtdIns4P is required for proper phagosomal acidification. Defective acidification may be caused by impaired recruitment of Rab7 effectors, including RILP, which were shown earlier to displace phagosomes toward perinuclear lysosomes. Our results show multimodal dynamics of PtdIns4P during phagocytosis and suggest that the phosphoinositide plays important roles during the maturation of the phagosome. PMID:28035045

  8. PI4P5-kinase Ialpha is required for efficient HIV-1 entry and infection of T cells.

    PubMed

    Barrero-Villar, Marta; Barroso-González, Jonathan; Cabrero, J R; Gordón-Alonso, Mónica; Alvarez-Losada, Susana; Muñoz-Fernández, M A; Sánchez-Madrid, Francisco; Valenzuela-Fernández, Agustín

    2008-11-15

    HIV-1 envelope (Env) triggers membrane fusion between the virus and the target cell. The cellular mechanism underlying this process is not well known. Phosphatidylinositol 4,5-bisphosphate (PIP(2)) is known to be important for the late steps of the HIV-1 infection cycle by promoting Gag localization to the plasma membrane during viral assembly, but it has not been implicated in early stages of HIV-1 membrane-related events. In this study, we show that binding of the initial HIV-1 Env-gp120 protein induces PIP(2) production in permissive lymphocytes through the activation of phosphatidylinositol-4-phosphate 5-kinase (PI4P5-K) Ialpha. Overexpression of wild-type PI4P5-K Ialpha increased HIV-1 Env-mediated PIP(2) production and enhanced viral replication in primary lymphocytes and CEM T cells, whereas PIP(2) production and HIV-1 infection were both severely reduced in cells overexpressing the kinase-dead mutant D227A (D/A)-PI4P5-K Ialpha. Similar results were obtained with replicative and single-cycle HIV-1 particles. HIV-1 infection was also inhibited by knockdown of endogenous expression of PI4P5-K Ialpha. These data indicate that PI4P5-K Ialpha-mediated PIP(2) production is crucial for HIV-1 entry and the early steps of infection in permissive lymphocytes.

  9. Arabidopsis D6PK is a lipid domain-dependent mediator of root epidermal planar polarity.

    PubMed

    Stanislas, Thomas; Hüser, Anke; Barbosa, Inês C R; Kiefer, Christian S; Brackmann, Klaus; Pietra, Stefano; Gustavsson, Anna; Zourelidou, Melina; Schwechheimer, Claus; Grebe, Markus

    2015-11-02

    Development of diverse multicellular organisms relies on coordination of single-cell polarities within the plane of the tissue layer (planar polarity). Cell polarity often involves plasma membrane heterogeneity generated by accumulation of specific lipids and proteins into membrane subdomains. Coordinated hair positioning along Arabidopsis root epidermal cells provides a planar polarity model in plants, but knowledge about the functions of proteo-lipid domains in planar polarity signalling remains limited. Here we show that Rho-of-plant (ROP) 2 and 6, phosphatidylinositol-4-phosphate 5-kinase 3 (PIP5K3), DYNAMIN-RELATED PROTEIN (DRP) 1A and DRP2B accumulate in a sterol-enriched, polar membrane domain during root hair initiation. DRP1A, DRP2B, PIP5K3 and sterols are required for planar polarity and the AGCVIII kinase D6 PROTEIN KINASE (D6PK) is a modulator of this process. D6PK undergoes phosphatidylinositol-4,5-bisphosphate- and sterol-dependent basal-to-planar polarity switching into the polar, lipid-enriched domain just before hair formation, unravelling lipid-dependent D6PK localization during late planar polarity signalling.

  10. Phosphatidylinositol(4,5)bisphosphate and phosphatidylinositol(4)phosphate in plant tissues. [Pisum sativum

    SciTech Connect

    Irvine, R.F.; Letcher, A.J.; Lander, D.J. ); Dawson, A.P. ); Musgrave, A. ); Drobak, B.K. )

    1989-03-01

    Pea (Pisum sativum) leaf discs or swimming suspensions of Chlamydomonas eugametos were radiolabeled with ({sup 3}H)myo-inositol or ({sup 32}P)Pi and the lipids were extracted, deacylated, and their glycerol moieties removed. The resulting inositol trisphosphate and bisphosphate fractions were examined by periodate degradation, reduction and dephosphorylation, or by incubation with human red cell membranes. Their likely structures were identified as D-myo-inositol(1,4,5)trisphosphate and D-myo-inositol(1,4,)-bisphosphate. It is concluded that plants contain phosphatidylinositol(4)phosphate and phosphatidylinositol(4,5)bisphosphate; no other polyphosphoinositides were detected.

  11. Erythritol feeds the pentose phosphate pathway via three new isomerases leading to D-erythrose-4-phosphate in Brucella

    PubMed Central

    Barbier, Thibault; Collard, François; Zúñiga-Ripa, Amaia; Moriyón, Ignacio; Godard, Thibault; Becker, Judith; Wittmann, Christoph; Van Schaftingen, Emile; Letesson, Jean-Jacques

    2014-01-01

    Erythritol is an important nutrient for several α-2 Proteobacteria, including N2-fixing plant endosymbionts and Brucella, a worldwide pathogen that finds this four-carbon polyol in genital tissues. Erythritol metabolism involves phosphorylation to l-erythritol-4-phosphate by the kinase EryA and oxidation of the latter to l-3-tetrulose 4-phosphate by the dehydrogenase EryB. It is accepted that further steps involve oxidation by the putative dehydrogenase EryC and subsequent decarboxylation to yield triose-phosphates. Accordingly, growth on erythritol as the sole C source should require aldolase and fructose-1,6-bisphosphatase to produce essential hexose-6-monophosphate. However, we observed that a mutant devoid of fructose-1,6-bisphosphatases grew normally on erythritol and that EryC, which was assumed to be a dehydrogenase, actually belongs to the xylose isomerase superfamily. Moreover, we found that TpiA2 and RpiB, distant homologs of triose phosphate isomerase and ribose 5-phosphate isomerase B, were necessary, as previously shown for Rhizobium. By using purified recombinant enzymes, we demonstrated that l-3-tetrulose-4-phosphate was converted to d-erythrose 4-phosphate through three previously unknown isomerization reactions catalyzed by EryC (tetrulose-4-phosphate racemase), TpiA2 (d-3-tetrulose-4-phosphate isomerase; renamed EryH), and RpiB (d-erythrose-4-phosphate isomerase; renamed EryI), a pathway fully consistent with the isotopomer distribution of the erythrose-4-phosphate-derived amino acids phenylalanine and tyrosine obtained from bacteria grown on 13C-labeled erythritol. d-Erythrose-4-phosphate is then converted by enzymes of the pentose phosphate pathway to glyceraldehyde 3-phosphate and fructose 6-phosphate, thus bypassing fructose-1,6-bisphosphatase. This is the first description to our knowledge of a route feeding carbohydrate metabolism exclusively via d-erythrose 4-phosphate, a pathway that may provide clues to the preferential metabolism of

  12. Erythritol feeds the pentose phosphate pathway via three new isomerases leading to D-erythrose-4-phosphate in Brucella.

    PubMed

    Barbier, Thibault; Collard, François; Zúñiga-Ripa, Amaia; Moriyón, Ignacio; Godard, Thibault; Becker, Judith; Wittmann, Christoph; Van Schaftingen, Emile; Letesson, Jean-Jacques

    2014-12-16

    Erythritol is an important nutrient for several α-2 Proteobacteria, including N2-fixing plant endosymbionts and Brucella, a worldwide pathogen that finds this four-carbon polyol in genital tissues. Erythritol metabolism involves phosphorylation to L-erythritol-4-phosphate by the kinase EryA and oxidation of the latter to L-3-tetrulose 4-phosphate by the dehydrogenase EryB. It is accepted that further steps involve oxidation by the putative dehydrogenase EryC and subsequent decarboxylation to yield triose-phosphates. Accordingly, growth on erythritol as the sole C source should require aldolase and fructose-1,6-bisphosphatase to produce essential hexose-6-monophosphate. However, we observed that a mutant devoid of fructose-1,6-bisphosphatases grew normally on erythritol and that EryC, which was assumed to be a dehydrogenase, actually belongs to the xylose isomerase superfamily. Moreover, we found that TpiA2 and RpiB, distant homologs of triose phosphate isomerase and ribose 5-phosphate isomerase B, were necessary, as previously shown for Rhizobium. By using purified recombinant enzymes, we demonstrated that L-3-tetrulose-4-phosphate was converted to D-erythrose 4-phosphate through three previously unknown isomerization reactions catalyzed by EryC (tetrulose-4-phosphate racemase), TpiA2 (D-3-tetrulose-4-phosphate isomerase; renamed EryH), and RpiB (D-erythrose-4-phosphate isomerase; renamed EryI), a pathway fully consistent with the isotopomer distribution of the erythrose-4-phosphate-derived amino acids phenylalanine and tyrosine obtained from bacteria grown on (13)C-labeled erythritol. D-erythrose-4-phosphate is then converted by enzymes of the pentose phosphate pathway to glyceraldehyde 3-phosphate and fructose 6-phosphate, thus bypassing fructose-1,6-bisphosphatase. This is the first description to our knowledge of a route feeding carbohydrate metabolism exclusively via D-erythrose 4-phosphate, a pathway that may provide clues to the preferential metabolism of

  13. Histones Cause Aggregation and Fusion of Lipid Vesicles Containing Phosphatidylinositol-4-Phosphate

    PubMed Central

    Lete, Marta G.; Sot, Jesus; Gil, David; Valle, Mikel; Medina, Milagros; Goñi, Felix M.; Alonso, Alicia

    2015-01-01

    In a previous article, we demonstrated that histones (H1 or histone octamers) interact with negatively charged bilayers and induce extensive aggregation of vesicles containing phosphatidylinositol-4-phosphate (PIP) and, to a lesser extent, vesicles containing phosphatidylinositol (PI). Here, we found that vesicles containing PIP, but not those containing PI, can undergo fusion induced by histones. Fusion was demonstrated through the observation of intervesicular mixing of total lipids and inner monolayer lipids, and by ultrastructural and confocal microscopy studies. Moreover, in both PI- and PIP-containing vesicles, histones caused permeabilization and release of vesicular aqueous contents, but the leakage mechanism was different (all-or-none for PI and graded release for PIP vesicles). These results indicate that histones could play a role in the remodeling of the nuclear envelope that takes place during the mitotic cycle. PMID:25692591

  14. Histones cause aggregation and fusion of lipid vesicles containing phosphatidylinositol-4-phosphate.

    PubMed

    Lete, Marta G; Sot, Jesus; Gil, David; Valle, Mikel; Medina, Milagros; Goñi, Felix M; Alonso, Alicia

    2015-02-17

    In a previous article, we demonstrated that histones (H1 or histone octamers) interact with negatively charged bilayers and induce extensive aggregation of vesicles containing phosphatidylinositol-4-phosphate (PIP) and, to a lesser extent, vesicles containing phosphatidylinositol (PI). Here, we found that vesicles containing PIP, but not those containing PI, can undergo fusion induced by histones. Fusion was demonstrated through the observation of intervesicular mixing of total lipids and inner monolayer lipids, and by ultrastructural and confocal microscopy studies. Moreover, in both PI- and PIP-containing vesicles, histones caused permeabilization and release of vesicular aqueous contents, but the leakage mechanism was different (all-or-none for PI and graded release for PIP vesicles). These results indicate that histones could play a role in the remodeling of the nuclear envelope that takes place during the mitotic cycle.

  15. Combretastatin A4-phosphate and its potential in veterinary oncology: a review.

    PubMed

    Abma, E; Daminet, S; Smets, P; Ni, Y; de Rooster, H

    2017-03-01

    For many years, research on anticancer therapy has focussed almost exclusively on targeting cancer cells directly, to selectively kill them or restrict their growth. But limited advances in this strategy have led researchers to shift their attention to other potential targets. Active research is now on-going on targeting tumour stroma. Vascular disrupting agents (VDAs) appear a promising class of anticancer drugs that are currently under investigation as a sole or combined therapy in human cancer patients. This article will briefly touch on the history and biology of combretastatin A4-phosphate (CA4P) as a typical example of VDAs and will concentrate on the side effects that can be expected when used in veterinary patients. Particularly, the pathogenesis of these side effects and how they may be prevented and/or treated will be discussed. The purpose of this article is to illustrate the potentials of CA4P as anticancer therapy in veterinary oncology patients.

  16. Contribution of PIP-5 kinase I{alpha} to raft-based Fc{gamma}RIIA signaling

    SciTech Connect

    Szymanska, Ewelina; Korzeniowski, Marek; Raynal, Patrick; Sobota, Andrzej; Kwiatkowska, Katarzyna

    2009-04-01

    Receptor Fc{gamma}IIA (Fc{gamma}RIIA) associates with plasma membrane rafts upon activation to trigger signaling cascades leading to actin polymerization. We examined whether compartmentalization of PI(4,5)P{sub 2} and PI(4,5)P{sub 2}-synthesizing PIP5-kinase I{alpha} to rafts contributes to Fc{gamma}RIIA signaling. A fraction of PIP5-kinase I{alpha} was detected in raft-originating detergent-resistant membranes (DRM) isolated from U937 monocytes and other cells. The DRM of U937 monocytes contained also a major fraction of PI(4,5)P{sub 2}. PIP5-kinase I{alpha} bound PI(4,5)P{sub 2}, and depletion of the lipid displaced PIP5-kinase I{alpha} from the DRM. Activation of Fc{gamma}RIIA in BHK transfectants led to recruitment of the kinase to the plasma membrane and enrichment of DRM in PI(4,5)P{sub 2}. Immunofluorescence studies revealed that in resting cells the kinase was associated with the plasma membrane, cytoplasmic vesicles and the nucleus. After Fc{gamma}RIIA activation, PIP5-kinase I{alpha} and PI(4,5)P{sub 2} co-localized transiently with the activated receptor at distinct cellular locations. Immunoelectron microscopy studies revealed that PIP5-kinase I{alpha} and PI(4,5)P{sub 2} were present at the edges of electron-dense assemblies containing activated Fc{gamma}RIIA in their core. The data suggest that activation of Fc{gamma}RIIA leads to membrane rafts coalescing into signaling platforms containing PIP5-kinase I{alpha} and PI(4,5)P{sub 2}.

  17. Osh4p exchanges sterols for phosphatidylinositol 4-phosphate between lipid bilayers

    PubMed Central

    de Saint-Jean, Maud; Delfosse, Vanessa; Douguet, Dominique; Chicanne, Gaëtan; Payrastre, Bernard; Bourguet, William

    2011-01-01

    Osh/Orp proteins transport sterols between organelles and are involved in phosphoinositide metabolism. The link between these two aspects remains elusive. Using novel assays, we address the influence of membrane composition on the ability of Osh4p/Kes1p to extract, deliver, or transport dehydroergosterol (DHE). Surprisingly, phosphatidylinositol 4-phosphate (PI(4)P) specifically inhibited DHE extraction because PI(4)P was itself efficiently extracted by Osh4p. We solve the structure of the Osh4p–PI(4)P complex and reveal how Osh4p selectively substitutes PI(4)P for sterol. Last, we show that Osh4p quickly exchanges DHE for PI(4)P and, thereby, can transport these two lipids between membranes along opposite routes. These results suggest a model in which Osh4p transports sterol from the ER to late compartments pinpointed by PI(4)P and, in turn, transports PI(4)P backward. Coupled to PI(4)P metabolism, this transport cycle would create sterol gradients. Because the residues that recognize PI(4)P are conserved in Osh4p homologues, other Osh/Orp are potential sterol/phosphoinositol phosphate exchangers. PMID:22162133

  18. INTRACELLULAR TRANSPORT. Phosphatidylserine transport by ORP/Osh proteins is driven by phosphatidylinositol 4-phosphate.

    PubMed

    Moser von Filseck, Joachim; Čopič, Alenka; Delfosse, Vanessa; Vanni, Stefano; Jackson, Catherine L; Bourguet, William; Drin, Guillaume

    2015-07-24

    In eukaryotic cells, phosphatidylserine (PS) is synthesized in the endoplasmic reticulum (ER) but is highly enriched in the plasma membrane (PM), where it contributes negative charge and to specific recruitment of signaling proteins. This distribution relies on transport mechanisms whose nature remains elusive. Here, we found that the PS transporter Osh6p extracted phosphatidylinositol 4-phosphate (PI4P) and exchanged PS for PI4P between two membranes. We solved the crystal structure of Osh6p:PI4P complex and demonstrated that the transport of PS by Osh6p depends on PI4P recognition in vivo. Finally, we showed that the PI4P-phosphatase Sac1p, by maintaining a PI4P gradient at the ER/PM interface, drove PS transport. Thus, PS transport by oxysterol-binding protein-related protein (ORP)/oxysterol-binding homology (Osh) proteins is fueled by PI4P metabolism through PS/PI4P exchange cycles.

  19. Characterizing the Tumor Response to Treatment With Combretastatin A4 Phosphate

    SciTech Connect

    Salmon, Beth A.; Siemann, Dietmar W. . E-mail: siemadw@ufl.edu

    2007-05-01

    Purpose: To examine the pathophysiologic impact of treatment with combretastatin A4 phosphate (CA4P) in regions of tumors that ultimately either necrose or survive treatment with this agent. Methods and Materials: Proliferation, perfusion, vessel density, and expression of vascular endothelial growth factor (VEGF) were analyzed in the KHT tumor model after treatment with CA4P. Analyses were conducted in the whole tumor and the tumor periphery. Results: Perfusion in the tumor periphery decreased 4 h after treatment, but returned to baseline 20 h later. Whole-tumor perfusion also decreased 4 h after treatment, but did not return to baseline. Vessel density decreased in the tumor as a whole, but not in the tumor periphery. No significant effect on the expression of VEGF was observed, but a decrease in proliferation in the whole tumor and the periphery was noted. Conclusions: The present study shows that those areas of a tumor that survive treatment with CA4P are affected by CA4P exposure, though only transiently. The decrease in perfusion could negatively affect therapies utilizing the combination of CA4P and conventional anticancer agents by decreasing drug delivery and tissue oxygenation. These findings suggest that the timing of CA4P treatments when used in conjunction with conventional anticancer therapies should be considered carefully.

  20. Endosomal type Iγ PIP 5-kinase controls EGF receptor lysosomal sorting.

    PubMed

    Sun, Yue; Hedman, Andrew C; Tan, Xiaojun; Schill, Nicholas J; Anderson, Richard A

    2013-04-29

    Endosomal trafficking and degradation of epidermal growth factor receptor (EGFR) play an essential role in the control of its signaling. Phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P(2)) is an established regulator of endocytosis, whereas PtdIns3P modulates endosomal trafficking. However, we demonstrate here that type I gamma phosphatidylinositol phosphate 5-kinase i5 (PIPKIγi5), an enzyme that synthesizes PtdIns4,5P(2), controls endosome-to-lysosome sorting of EGFR. In this pathway, PIPKIγi5 interacts with sorting nexin 5 (SNX5), a protein that binds PtdIns4,5P(2) and other phosphoinositides. PIPKIγi5 and SNX5 localize to endosomes, and loss of either protein blocks EGFR sorting into intraluminal vesicles (ILVs) of the multivesicular body. Loss of ILV sorting greatly enhances and prolongs EGFR signaling. PIPKIγi5 and SNX5 prevent Hrs ubiquitination, and this facilitates the Hrs association with EGFR that is required for ILV sorting. These findings reveal that PIPKIγi5 and SNX5 form a signaling nexus that controls EGFR endosomal sorting, degradation, and signaling.

  1. Feedback inhibition of deoxy-D-xylulose-5-phosphate synthase regulates the methylerythritol 4-phosphate pathway.

    PubMed

    Banerjee, Aparajita; Wu, Yan; Banerjee, Rahul; Li, Yue; Yan, Honggao; Sharkey, Thomas D

    2013-06-07

    The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway leads to the biosynthesis of isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), the precursors for isoprene and higher isoprenoids. Isoprene has significant effects on atmospheric chemistry, whereas other isoprenoids have diverse roles ranging from various biological processes to applications in commercial uses. Understanding the metabolic regulation of the MEP pathway is important considering the numerous applications of this pathway. The 1-deoxy-D-xylulose-5-phosphate synthase (DXS) enzyme was cloned from Populus trichocarpa, and the recombinant protein (PtDXS) was purified from Escherichia coli. The steady-state kinetic parameters were measured by a coupled enzyme assay. An LC-MS/MS-based assay involving the direct quantification of the end product of the enzymatic reaction, 1-deoxy-D-xylulose 5-phosphate (DXP), was developed. The effect of different metabolites of the MEP pathway on PtDXS activity was tested. PtDXS was inhibited by IDP and DMADP. Both of these metabolites compete with thiamine pyrophosphate for binding with the enzyme. An atomic structural model of PtDXS in complex with thiamine pyrophosphate and Mg(2+) was built by homology modeling and refined by molecular dynamics simulations. The refined structure was used to model the binding of IDP and DMADP and indicated that IDP and DMADP might bind with the enzyme in a manner very similar to the binding of thiamine pyrophosphate. The feedback inhibition of PtDXS by IDP and DMADP constitutes an important mechanism of metabolic regulation of the MEP pathway and indicates that thiamine pyrophosphate-dependent enzymes may often be affected by IDP and DMADP.

  2. An efficient synthetic strategy for obtaining 4-methoxy carbon isotope labeled combretastatin A-4 phosphate and other Z-combretastatins.

    PubMed

    Pettit, George R; Minardi, Mathew D; Hogan, Fiona; Price, Pat M

    2010-03-26

    Human cancer and other clinical trials under development employing combretastatin A-4 phosphate (1b, CA4P) should benefit from the availability of a [(11)C]-labeled derivative for positron emission tomography (PET). In order to obtain a suitable precursor for addition of a [(11)C]methyl group at the penultimate step, several new synthetic pathways to CA4P were evaluated. Geometrical isomerization (Z to E) proved to be a challenge, but it was overcome by development of a new CA4P synthesis suitable for 4-methoxy isotope labeling.

  3. The Mycobacterium tuberculosis MEP (2C-methyl-D-erythritol 4-phosphate) pathway as a new drug target

    PubMed Central

    Eoh, Hyungjin; Brennan, Patrick J.; Crick, Dean C.

    2009-01-01

    Tuberculosis (TB) is still a major public health problem, compounded by the human immunodeficiency virus (HIV)-TB co-infection and recent emergence of multidrug-resistant (MDR) and extensive drug resistant (XDR)-TB. Novel anti-TB drugs are urgently required. In this context, the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway of Mycobacterium tuberculosis has drawn attention; it is one of several pathways vital for M. tuberculosis viability and the human host lacks homologous enzymes. Thus, the MEP pathway promises bacterium-specific drug targets and the potential for identification of lead compounds unencumbered by target-based toxicity. Indeed, fosmidomycin is now known to inhibit the second step in the MEP pathway. This review describes the cardinal features of the main enzymes of the MEP pathway in M. tuberculosis and how these can be manipulated in high throughput screening campaigns in the search for new anti-infectives against TB. PMID:18793870

  4. TIE2-expressing macrophages limit the therapeutic efficacy of the vascular-disrupting agent combretastatin A4 phosphate in mice

    PubMed Central

    Welford, Abigail F.; Biziato, Daniela; Coffelt, Seth B.; Nucera, Silvia; Fisher, Matthew; Pucci, Ferdinando; Di Serio, Clelia; Naldini, Luigi; De Palma, Michele; Tozer, Gillian M.; Lewis, Claire E.

    2011-01-01

    Vascular-disrupting agents (VDAs) such as combretastatin A4 phosphate (CA4P) selectively disrupt blood vessels in tumors and induce tumor necrosis. However, tumors rapidly repopulate after treatment with such compounds. Here, we show that CA4P-induced vessel narrowing, hypoxia, and hemorrhagic necrosis in murine mammary tumors were accompanied by elevated tumor levels of the chemokine CXCL12 and infiltration by proangiogenic TIE2-expressing macrophages (TEMs). Inhibiting TEM recruitment to CA4P-treated tumors either by interfering pharmacologically with the CXCL12/CXCR4 axis or by genetically depleting TEMs in tumor-bearing mice markedly increased the efficacy of CA4P treatment. These data suggest that TEMs limit VDA-induced tumor injury and represent a potential target for improving the clinical efficacy of VDA-based therapies. PMID:21490397

  5. TIE2-expressing macrophages limit the therapeutic efficacy of the vascular-disrupting agent combretastatin A4 phosphate in mice.

    PubMed

    Welford, Abigail F; Biziato, Daniela; Coffelt, Seth B; Nucera, Silvia; Fisher, Matthew; Pucci, Ferdinando; Di Serio, Clelia; Naldini, Luigi; De Palma, Michele; Tozer, Gillian M; Lewis, Claire E

    2011-05-01

    Vascular-disrupting agents (VDAs) such as combretastatin A4 phosphate (CA4P) selectively disrupt blood vessels in tumors and induce tumor necrosis. However, tumors rapidly repopulate after treatment with such compounds. Here, we show that CA4P-induced vessel narrowing, hypoxia, and hemorrhagic necrosis in murine mammary tumors were accompanied by elevated tumor levels of the chemokine CXCL12 and infiltration by proangiogenic TIE2-expressing macrophages (TEMs). Inhibiting TEM recruitment to CA4P-treated tumors either by interfering pharmacologically with the CXCL12/CXCR4 axis or by genetically depleting TEMs in tumor-bearing mice markedly increased the efficacy of CA4P treatment. These data suggest that TEMs limit VDA-induced tumor injury and represent a potential target for improving the clinical efficacy of VDA-based therapies.

  6. Plasmodium IspD (2-C-Methyl-D-erythritol 4-Phosphate Cytidyltransferase), an Essential and Druggable Antimalarial Target

    PubMed Central

    Imlay, Leah S.; Armstrong, Christopher M.; Masters, Mary Clare; Li, Ting; Price, Kathryn E.; Edwards, Rachel L.; Mann, Katherine M.; Li, Lucy X.; Stallings, Christina L.; Berry, Neil G.; O’Neill, Paul M.; Odom, Audrey R.

    2015-01-01

    As resistance to current therapies spreads, novel antimalarials are urgently needed. In this work, we examine the potential for therapeutic intervention via the targeting of Plasmodium IspD (2-C-methyl-D-erythritol 4-phosphate cytidyltransferase), the second dedicated enzyme of the essential methylerythritol phosphate (MEP) pathway for isoprenoid biosynthesis. Enzymes of this pathway represent promising therapeutic targets because the pathway is not present in humans. The Malaria Box compound, MMV008138, inhibits Plasmodium falciparum growth, and PfIspD has been proposed as a candidate intracellular target. We find that PfIspD is the sole intracellular target of MMV008138 and characterize the mode of inhibition and target-based resistance, providing chemical validation of this target. Additionally, we find that the Pf ISPD genetic locus is refractory to disruption in malaria parasites, providing independent genetic validation for efforts targeting this enzyme. This work provides compelling support for IspD as a druggable target for the development of additional, much-needed antimalarial agents. PMID:26783558

  7. [Cloning and expression analysis of 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase gene in Tripterygium wilfordii].

    PubMed

    Tong, Yu-ru; Su, Ping; Zhang, Meng; Zhao, Yu-jun; Wang, Xiu-juan; Gao, Wei; Huang, Lu-qi

    2015-11-01

    To clone the 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (TwMCT) full length cDNA from Tripterygium wilfordii, the specific primers were designed according to the transcriptome data and the LCPCR were carried out. After a series of bioinformatics analysis on the TwMCT, the MeJA induced expression content were investigated by real-time fluorescence quantification polymerase chain reaction (RT-qPCR). The result showed that the full of TwMCTcDNA was 1 318 bp nucleotides encoding 311 amino acids. The molecular weight of the deduced TwMCT protein was about 34.14 kDa and the theoretical isoelectric point was 8.65. Result of the RT-qPCR analysis indicated that the content of TwMCT mRNA expression in T. wilfordii suspension cell was rising after treating with MeJA and reached the maximum in 24 h. Cloning and analyzing TwMCT gene from T. wilfordii provided gene element for studying the function and expression regulation of secondary metabolites.

  8. GOLPH3 Bridges Phosphatidylinositol-4- Phosphate and Actomyosin to Stretch and Shape the Golgi to Promote Budding

    PubMed Central

    Dippold, Holly C.; Ng, Michelle M.; Farber-Katz, Suzette E.; Lee, Sun-Kyung; Kerr, Monica L.; Peterman, Marshall C.; Sim, Ronald; Wiharto, Patricia A.; Galbraith, Kenneth A.; Madhavarapu, Swetha; Fuchs, Greg J.; Meerloo, Timo; Farquhar, Marilyn G.; Zhou, Huilin; Field, Seth J.

    2009-01-01

    SUMMARY Golgi membranes, from yeast to humans, are uniquely enriched in phosphatidylinositol-4-phosphate (PtdIns(4)P), although the role of this lipid remains poorly understood. Using a proteomic lipid binding screen, we identify the Golgi protein GOLPH3 (also called GPP34, GMx33, MIDAS, or yeast Vps74p) as a PtdIns(4)P-binding protein that depends upon PtdIns(4)P for its Golgi localization. We further show that GOLPH3 binds the unconventional myosin MYO18A, thus connecting the Golgi to F-actin. We demonstrate that this linkage is necessary for normal Golgi trafficking and morphology. The evidence suggests that GOLPH3 binds to PtdIns(4)P-rich trans-Golgi membranes and MYO18A conveying a tensile force required for efficient tubule and vesicle formation. Consequently, this tensile force stretches the Golgi into the extended ribbon observed by fluorescence microscopy and the familiar flattened form observed by electron microscopy. PMID:19837035

  9. Phosphatidylinositol 4-phosphate in the Golgi apparatus regulates cell-cell adhesion and invasive cell migration in human breast cancer.

    PubMed

    Tokuda, Emi; Itoh, Toshiki; Hasegawa, Junya; Ijuin, Takeshi; Takeuchi, Yukiko; Irino, Yasuhiro; Fukumoto, Miki; Takenawa, Tadaomi

    2014-06-01

    Downregulation of cell-cell adhesion and upregulation of cell migration play critical roles in the conversion of benign tumors to aggressive invasive cancers. In this study, we show that changes in cell-cell adhesion and cancer cell migration/invasion capacity depend on the level of phosphatidylinositol 4-phosphate [PI(4)P] in the Golgi apparatus in breast cancer cells. Attenuating SAC1, a PI(4)P phosphatase localized in the Golgi apparatus, resulted in decreased cell-cell adhesion and increased cell migration in weakly invasive cells. In contrast, silencing phosphatidylinositol 4-kinase IIIβ, which generates PI(4)P in the Golgi apparatus, increased cell-cell adhesion and decreased invasion in highly invasive cells. Furthermore, a PI(4)P effector, Golgi phosphoprotein 3, was found to be involved in the generation of these phenotypes in a manner that depends on its PI(4)P-binding ability. Our results provide a new model for breast cancer cell progression in which progression is controlled by PI(4)P levels in the Golgi apparatus.

  10. Novel bioassay for the discovery of inhibitors of the 2-C-Methyl-D-Erythritol 4-Phosphate (MEP) and terpenoid pathways leading to carotenoid biosynthesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway leads to the synthesis of isopentenyl-phosphate (IPP) in plastids. It is a major branch point providing precursors for the synthesis of carotenoids, tocopherols, plastoquinone and the phytyl chain of chlorophylls, as well as the hormones abscisi...

  11. Molecular basis of phosphatidylinositol 4-phosphate and ARF1 GTPase recognition by the FAPP1 pleckstrin homology (PH) domain.

    PubMed

    He, Ju; Scott, Jordan L; Heroux, Annie; Roy, Siddhartha; Lenoir, Marc; Overduin, Michael; Stahelin, Robert V; Kutateladze, Tatiana G

    2011-05-27

    Four-phosphate-adaptor protein 1 (FAPP1) regulates secretory transport from the trans-Golgi network (TGN) to the plasma membrane. FAPP1 is recruited to the Golgi through binding of its pleckstrin homology (PH) domain to phosphatidylinositol 4-phosphate (PtdIns(4)P) and a small GTPase ADP-ribosylation factor 1 (ARF1). Despite the critical role of FAPP1 in membrane trafficking, the molecular basis of its dual function remains unclear. Here, we report a 1.9 Å resolution crystal structure of the FAPP1 PH domain and detail the molecular mechanisms of the PtdIns(4)P and ARF1 recognition. The FAPP1 PH domain folds into a seven-stranded β-barrel capped by an α-helix at one edge, whereas the opposite edge is flanked by three loops and the β4 and β7 strands that form a lipid-binding pocket within the β-barrel. The ARF1-binding site is located on the outer side of the β-barrel as determined by NMR resonance perturbation analysis, mutagenesis, and measurements of binding affinities. The two binding sites have little overlap, allowing FAPP1 PH to associate with both ligands simultaneously and independently. Binding to PtdIns(4)P is enhanced in an acidic environment and is required for membrane penetration and tubulation activity of FAPP1, whereas the GTP-bound conformation of the GTPase is necessary for the interaction with ARF1. Together, these findings provide structural and biochemical insight into the multivalent membrane anchoring by the PH domain that may augment affinity and selectivity of FAPP1 toward the TGN membranes enriched in both PtdIns(4)P and GTP-bound ARF1.

  12. Synthesis of unsaturated phosphatidylinositol 4-phosphates and the effects of substrate unsaturation on SopB phosphatase activity.

    PubMed

    Furse, Samuel; Mak, LokHang; Tate, Edward W; Templer, Richard H; Ces, Oscar; Woscholski, Rüdiger; Gaffney, Piers R J

    2015-02-21

    In this paper evidence is presented that the fatty acid component of an inositide substrate affects the kinetic parameters of the lipid phosphatase Salmonella Outer Protein B (SopB). A succinct route was used to prepare the naturally occurring enantiomer of phosphatidylinositol 4-phosphate (PI-4-P) with saturated, as well as singly, triply and quadruply unsaturated, fatty acid esters, in four stages: (1) The enantiomers of 2,3:5,6-O-dicyclohexylidene-myo-inositol were resolved by crystallisation of their di(acetylmandelate) diastereoisomers. (2) The resulting diol was phosphorylated regio-selectively exclusively on the 1-O using the new reagent tri(2-cyanoethyl)phosphite. (3) With the 4-OH still unprotected, the glyceride was coupled using phosphate tri-ester methodology. (4) A final phosphorylation of the 4-O, followed by global deprotection under basic then acidic conditions, provided PI-4-P bearing a range of sn-1-stearoyl, sn-2-stearoyl, -oleoyl, -γ-linolenoyl and arachidonoyl, glycerides. Enzymological studies showed that the introduction of cis-unsaturated bonds has a measurable influence on the activity (relative Vmax) of SopB. Mono-unsaturated PI-4-P exhibited a five-fold higher activity, with a two-fold higher KM, over the saturated substrate, when presented in DOPC vesicles. Poly-unsaturated PI-4-P showed little further change with respect to the singly unsaturated species. This result, coupled with our previous report that saturated PI-4-P has much higher stored curvature elastic stress than PI, supports the hypothesis that the activity of inositide phosphatase SopB has a physical role in vivo.

  13. Local control of phosphatidylinositol 4-phosphate signaling in the Golgi apparatus by Vps74 and Sac1 phosphoinositide phosphatase

    PubMed Central

    Wood, Christopher S.; Hung, Chia-Sui; Huoh, Yu-San; Mousley, Carl J.; Stefan, Christopher J.; Bankaitis, Vytas; Ferguson, Kathryn M.; Burd, Christopher G.

    2012-01-01

    In the Golgi apparatus, lipid homeostasis pathways are coordinated with the biogenesis of cargo transport vesicles by phosphatidylinositol 4-kinases (PI4Ks) that produce phosphatidylinositol 4-phosphate (PtdIns4P), a signaling molecule that is recognized by downstream effector proteins. Quantitative analysis of the intra-Golgi distribution of a PtdIns4P reporter protein confirms that PtdIns4P is enriched on the trans-Golgi cisterna, but surprisingly, Vps74 (the orthologue of human GOLPH3), a PI4K effector required to maintain residence of a subset of Golgi proteins, is distributed with the opposite polarity, being most abundant on cis and medial cisternae. Vps74 binds directly to the catalytic domain of Sac1 (KD = 3.8 μM), the major PtdIns4P phosphatase in the cell, and PtdIns4P is elevated on medial Golgi cisternae in cells lacking Vps74 or Sac1, suggesting that Vps74 is a sensor of PtdIns4P level on medial Golgi cisternae that directs Sac1-mediated dephosphosphorylation of this pool of PtdIns4P. Consistent with the established role of Sac1 in the regulation of sphingolipid biosynthesis, complex sphingolipid homeostasis is perturbed in vps74Δ cells. Mutant cells lacking complex sphingolipid biosynthetic enzymes fail to properly maintain residence of a medial Golgi enzyme, and cells lacking Vps74 depend critically on complex sphingolipid biosynthesis for growth. The results establish additive roles of Vps74-mediated and sphingolipid-dependent sorting of Golgi residents. PMID:22553352

  14. Structural definition of the active site and catalytic mechanism of 3,4-dihydroxy-2-butanone-4-phosphate synthase.

    PubMed

    Liao, Der-Ing; Zheng, Ya-Jun; Viitanen, Paul V; Jordan, Douglas B

    2002-02-12

    X-ray crystal structures of L-3,4-dihydroxy-2-butanone-4-phosphate synthase from Magnaporthe grisea are reported for the E-SO(4)(2-), E-SO(4)(2-)-Mg(2+), E-SO(4)(2)(-)-Mn(2+), E-SO(4)(2)(-)-Mn(2+)-glycerol, and E-SO(4)(2)(-)-Zn(2+) complexes with resolutions that extend to 1.55, 0.98, 1.60, 1.16, and 1.00 A, respectively. Active-site residues of the homodimer are fully defined. The structures were used to model the substrate ribulose 5-phosphate in the active site with the phosphate group anchored at the sulfate site and the placement of the ribulose group guided by the glycerol site. The model includes two Mg(2+) cations that bind to the oxygen substituents of the C2, C3, C4, and phosphate groups of the substrate, the side chains of Glu37 and His153, and water molecules. The position of the metal cofactors and the substrate's phosphate group are further stabilized by an extensive hydrogen-bond and salt-bridge network. On the basis of their proximity to the substrate's reaction participants, the imidazole of an Asp99-His136 dyad from one subunit, the side chains of the Asp41, Cys66, and Glu174 residues from the other subunit, and Mg(2+)-activated water molecules are proposed to serve specific roles in the catalytic cycle as general acid-base functionalities. The model suggests that during the 1,2-shift step of the reaction, the substrate's C3 and C4 hydroxyl groups are cis to each other. A cis transition state is calculated to have an activation barrier that is 2 kcal/mol greater than that of the trans transition state in the absence of the enzyme.

  15. Structural definition of the active site and catalytic mechanism of 3,4-dihydroxy-2-butanone 4-phosphate synthase

    SciTech Connect

    Liao, D.-I.; Zheng, Y.-J.; Viitanen, P.V.; Jordan, D.B.

    2010-03-08

    X-ray crystal structures of L-3,4-dihydroxy-2-butanone-4-phosphate synthase from Magnaporthe grisea are reported for the E-SO{sub 4}{sup 2-}, E-{sub 4}{sup 2-}-Mg{sup 2+}, E-SO{sub 4}{sup 2-}-Mn{sup 2+}, E-SO{sub 4}{sup 2-}-Mn{sup 2+}-glycerol, and E-SO{sub 4}{sup 2-}-Zn{sup 2+} complexes with resolutions that extend to 1.55, 0.98, 1.60, 1.16, and 1.00 {angstrom}, respectively. Active-site residues of the homodimer are fully defined. The structures were used to model the substrate ribulose 5-phosphate in the active site with the phosphate group anchored at the sulfate site and the placement of the ribulose group guided by the glycerol site. The model includes two Mg{sup 2+} cations that bind to the oxygen substituents of the C2, C3, C4, and phosphate groups of the substrate, the side chains of Glu37 and His153, and water molecules. The position of the metal cofactors and the substrate's phosphate group are further stabilized by an extensive hydrogen-bond and salt-bridge network. On the basis of their proximity to the substrate's reaction participants, the imidazole of an Asp99-His136 dyad from one subunit, the side chains of the Asp41, Cys66, and Glu174 residues from the other subunit, and Mg{sup 2+}-activated water molecules are proposed to serve specific roles in the catalytic cycle as general acid-base functionalities. The model suggests that during the 1,2-shift step of the reaction, the substrate's C3 and C4 hydroxyl groups are cis to each other. A cis transition state is calculated to have an activation barrier that is 2 kcal/mol greater than that of the trans transition state in the absence of the enzyme.

  16. Eps15 homology domain 1-associated tubules contain phosphatidylinositol-4-phosphate and phosphatidylinositol-(4,5)-bisphosphate and are required for efficient recycling.

    PubMed

    Jović, Marko; Kieken, Fabien; Naslavsky, Naava; Sorgen, Paul L; Caplan, Steve

    2009-06-01

    The C-terminal Eps15 homology domain (EHD) 1/receptor-mediated endocytosis-1 protein regulates recycling of proteins and lipids from the recycling compartment to the plasma membrane. Recent studies have provided insight into the mode by which EHD1-associated tubular membranes are generated and the mechanisms by which EHD1 functions. Despite these advances, the physiological function of these striking EHD1-associated tubular membranes remains unknown. Nuclear magnetic resonance spectroscopy demonstrated that the Eps15 homology (EH) domain of EHD1 binds to phosphoinositides, including phosphatidylinositol-4-phosphate. Herein, we identify phosphatidylinositol-4-phosphate as an essential component of EHD1-associated tubules in vivo. Indeed, an EHD1 EH domain mutant (K483E) that associates exclusively with punctate membranes displayed decreased binding to phosphatidylinositol-4-phosphate and other phosphoinositides. Moreover, we provide evidence that although the tubular membranes to which EHD1 associates may be stabilized and/or enhanced by EHD1 expression, these membranes are, at least in part, pre-existing structures. Finally, to underscore the function of EHD1-containing tubules in vivo, we used a small interfering RNA (siRNA)/rescue assay. On transfection, wild-type, tubule-associated, siRNA-resistant EHD1 rescued transferrin and beta1 integrin recycling defects observed in EHD1-depleted cells, whereas expression of the EHD1 K483E mutant did not. We propose that phosphatidylinositol-4-phosphate is an essential component of EHD1-associated tubules that also contain phosphatidylinositol-(4,5)-bisphosphate and that these structures are required for efficient recycling to the plasma membrane.

  17. The Plastidial 2-C-Methyl-d-Erythritol 4-Phosphate Pathway Provides the Isoprenyl Moiety for Protein Geranylgeranylation in Tobacco BY-2 Cells[C][W

    PubMed Central

    Gerber, Esther; Hemmerlin, Andréa; Hartmann, Michael; Heintz, Dimitri; Hartmann, Marie-Andrée; Mutterer, Jérôme; Rodríguez-Concepción, Manuel; Boronat, Albert; Van Dorsselaer, Alain; Rohmer, Michel; Crowell, Dring N.; Bach, Thomas J.

    2009-01-01

    Protein farnesylation and geranylgeranylation are important posttranslational modifications in eukaryotic cells. We visualized in transformed Nicotiana tabacum Bright Yellow-2 (BY-2) cells the geranylgeranylation and plasma membrane localization of GFP-BD-CVIL, which consists of green fluorescent protein (GFP) fused to the C-terminal polybasic domain (BD) and CVIL isoprenylation motif from the Oryza sativa calmodulin, CaM61. Treatment with fosmidomycin (Fos) or oxoclomazone (OC), inhibitors of the plastidial 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, caused mislocalization of the protein to the nucleus, whereas treatment with mevinolin, an inhibitor of the cytosolic mevalonate pathway, did not. The nuclear localization of GFP-BD-CVIL in the presence of MEP pathway inhibitors was completely reversed by all-trans-geranylgeraniol (GGol). Furthermore, 1-deoxy-d-xylulose (DX) reversed the effects of OC, but not Fos, consistent with the hypothesis that OC blocks 1-deoxy-d-xylulose 5-phosphate synthesis, whereas Fos inhibits its conversion to 2-C-methyl-d-erythritol 4-phosphate. By contrast, GGol and DX did not rescue the nuclear mislocalization of GFP-BD-CVIL in the presence of a protein geranylgeranyltransferase type 1 inhibitor. Thus, the MEP pathway has an essential role in geranylgeranyl diphosphate (GGPP) biosynthesis and protein geranylgeranylation in BY-2 cells. GFP-BD-CVIL is a versatile tool for identifying pharmaceuticals and herbicides that interfere either with GGPP biosynthesis or with protein geranylgeranylation. PMID:19136647

  18. Kinetic analysis of Escherichia coli 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase, wild type and mutants, reveals roles of active site amino acids.

    PubMed

    Richard, Stéphane B; Lillo, Antonietta M; Tetzlaff, Charles N; Bowman, Marianne E; Noel, Joseph P; Cane, David E

    2004-09-28

    Escherichia coli 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase (YgbP or IspD) catalyzes the conversion of 2-C-methyl-D-erythritol 4-phosphate (MEP) and cytidine triphosphate (CTP) to 4-diphosphocytidyl-2-C-methylerythritol (CDPME). Pulse chase experiments established that the reaction involves an ordered sequential mechanism with mandatory initial binding of CTP. On the basis of analysis of the previously reported crystal structures of apo-YgbP as well as YgbP complexed with both CTP.Mg(2+) and CDPME.Mg(2+) [Richard, S. B., Bowman, M. E., Kwiatkowski, W., Kang, I., Chow, C., Lillo, A. M., Cane, D. E., and Noel, J. P. (2001) Nat. Struct. Biol. 8, 641-648], a group of active site residues were selected for site-directed mutagenesis and steady-state kinetic analysis. Both Lys27 and Lys213 were shown to be essential to catalytic activity, consistent with their proposed role in stabilization of a pentacoordinate phosphate transition state resulting from in-line attack of the MEP phosphate on the alpha-phosphate of CTP. In addition, Thr140, Arg109, Asp106, and Thr165 were all shown to play critical roles in the binding and proper orientation of the MEP substrate.

  19. Cloning and characterization of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway genes of a natural-rubber producing plant, Hevea brasiliensis.

    PubMed

    Sando, Tomoki; Takeno, Shinya; Watanabe, Norie; Okumoto, Hiroshi; Kuzuyama, Tomohisa; Yamashita, Atsushi; Hattori, Masahira; Ogasawara, Naotake; Fukusaki, Eiichiro; Kobayashi, Akio

    2008-11-01

    Natural rubber is synthesized as rubber particles in the latex, the fluid cytoplasm of laticifers, of Hevea brasiliensis. Although it has been found that natural rubber is biosynthesized through the mevalonate pathway, the involvement of an alternative 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is uncertain. We obtained all series of the MEP pathway candidate genes by analyzing expressed sequence tag (EST) information and degenerate PCR in H. brasiliensis. Complementation experiments with Escherichia coli mutants were performed to confirm the functions of the MEP pathway gene products of H. brasiliensis together with those of Arabidopsis thaliana, and it was found that 1-deoxy-D-xylulose-5-phosphate reductoisomerase, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase, and 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase of H. brasiliensis were functionally active in the E. coli mutants. Gene expression analysis revealed that the expression level of the HbDXS2 gene in latex was relatively high as compared to those of other MEP pathway genes. However, a feeding experiment with [1-(13)C] 1-deoxy-D-xylulose triacetate, an intermediate derivative of the MEP pathway, indicated that the MEP pathway is not involved in rubber biosynthesis, but is involved in carotenoids biosynthesis in H. brasiliensis.

  20. A structural and functional study on the 2-C-methyl-d-erythritol-4-phosphate cytidyltransferase (IspD) from Bacillus subtilis

    PubMed Central

    Jin, Yun; Liu, Zhongchuan; Li, Yanjie; Liu, Weifeng; Tao, Yong; Wang, Ganggang

    2016-01-01

    2-C-Methyl-D-erythritol-4-phosphate cytidyltransferase (IspD) is an essential enzyme in the mevalonate-independent pathway of isoprenoid biosynthesis. This enzyme catalyzes 2-C-Methyl-d-erythritol 4-phosphate (MEP) and cytosine triphosphate (CTP) to 4-diphosphocytidyl-2-C-methyl-d-erythritol (CDPME) and inorganic pyrophosphate (PPi). Bacillus subtilis was a kind of excellent isoprene producer. However, the studies on the key enzymes of MEP pathway in B. subtilis were still absent. In this work, the crystal structures of IspD and IspD complexed with CTP from B.subtilis were determined. For the first time, the intact P-loop was observed in the apo structure of IspD enzyme. Structural comparisons revealed that the concerted movements of the P-loop and loops close to the active site were essential in the reaction catalyzed by IspD. Meanwhile, kinetic analysis showed that the CTP hydrolytic activity of IspD from B.subtilis was over two times higher than that from Escherichia coli. These results will be useful for future target-based screening of potential inhibitors and the metabolic engineering for isoprenoid biosynthesis. PMID:27821871

  1. Inositol phosphate pathway controls transcription of telomeric expression sites in trypanosomes.

    PubMed

    Cestari, Igor; Stuart, Ken

    2015-05-26

    African trypanosomes evade clearance by host antibodies by periodically changing their variant surface glycoprotein (VSG) coat. They transcribe only one VSG gene at a time from 1 of about 20 telomeric expression sites (ESs). They undergo antigenic variation by switching transcription between telomeric ESs or by recombination of the VSG gene expressed. We show that the inositol phosphate (IP) pathway controls transcription of telomeric ESs and VSG antigenic switching in Trypanosoma brucei. Conditional knockdown of phosphatidylinositol 5-kinase (TbPIP5K) or phosphatidylinositol 5-phosphatase (TbPIP5Pase) or overexpression of phospholipase C (TbPLC) derepresses numerous silent ESs in T. brucei bloodstream forms. The derepression is specific to telomeric ESs, and it coincides with an increase in the number of colocalizing telomeric and RNA polymerase I foci in the nucleus. Monoallelic VSG transcription resumes after reexpression of TbPIP5K; however, most of the resultant cells switched the VSG gene expressed. TbPIP5K, TbPLC, their substrates, and products localize to the plasma membrane, whereas TbPIP5Pase localizes to the nucleus proximal to telomeres. TbPIP5Pase associates with repressor/activator protein 1 (TbRAP1), and their telomeric silencing function is altered by TbPIP5K knockdown. These results show that specific steps in the IP pathway control ES transcription and antigenic switching in T. brucei by epigenetic regulation of telomere silencing.

  2. Inositol phosphate pathway controls transcription of telomeric expression sites in trypanosomes

    PubMed Central

    Cestari, Igor; Stuart, Ken

    2015-01-01

    African trypanosomes evade clearance by host antibodies by periodically changing their variant surface glycoprotein (VSG) coat. They transcribe only one VSG gene at a time from 1 of about 20 telomeric expression sites (ESs). They undergo antigenic variation by switching transcription between telomeric ESs or by recombination of the VSG gene expressed. We show that the inositol phosphate (IP) pathway controls transcription of telomeric ESs and VSG antigenic switching in Trypanosoma brucei. Conditional knockdown of phosphatidylinositol 5-kinase (TbPIP5K) or phosphatidylinositol 5-phosphatase (TbPIP5Pase) or overexpression of phospholipase C (TbPLC) derepresses numerous silent ESs in T. brucei bloodstream forms. The derepression is specific to telomeric ESs, and it coincides with an increase in the number of colocalizing telomeric and RNA polymerase I foci in the nucleus. Monoallelic VSG transcription resumes after reexpression of TbPIP5K; however, most of the resultant cells switched the VSG gene expressed. TbPIP5K, TbPLC, their substrates, and products localize to the plasma membrane, whereas TbPIP5Pase localizes to the nucleus proximal to telomeres. TbPIP5Pase associates with repressor/activator protein 1 (TbRAP1), and their telomeric silencing function is altered by TbPIP5K knockdown. These results show that specific steps in the IP pathway control ES transcription and antigenic switching in T. brucei by epigenetic regulation of telomere silencing. PMID:25964327

  3. Development of inhibitors of the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway enzymes as potential anti-infective agents.

    PubMed

    Masini, Tiziana; Hirsch, Anna K H

    2014-12-11

    Important pathogens such as Mycobacterium tuberculosis and Plasmodium falciparum, the causative agents of tuberculosis and malaria, respectively, and plants, utilize the 2C-methyl-D-erythritol 4-phosphate (MEP, 5) pathway for the biosynthesis of isopentenyl diphosphate (1) and dimethylallyl diphosphate (2), the universal precursors of isoprenoids, while humans exclusively utilize the alternative mevalonate pathway for the synthesis of 1 and 2. This distinct distribution, together with the fact that the MEP pathway is essential in numerous organisms, makes the enzymes of the MEP pathway attractive drug targets for the development of anti-infective agents and herbicides. Herein, we review the inhibitors reported over the past 2 years, in the context of the most important older developments and with a particular focus on the results obtained against enzymes of pathogenic organisms. We will also discuss new discoveries in terms of structural and mechanistic features, which can help to guide a rational development of inhibitors.

  4. Identification of a major IP5 kinase in Cryptococcus neoformans confirms that PP-IP5/IP7, not IP6, is essential for virulence

    PubMed Central

    Li, Cecilia; Lev, Sophie; Saiardi, Adolfo; Desmarini, Desmarini; Sorrell, Tania C.; Djordjevic, Julianne T.

    2016-01-01

    Fungal inositol polyphosphate (IP) kinases catalyse phosphorylation of IP3 to inositol pyrophosphate, PP-IP5/IP7, which is essential for virulence of Cryptococcus neoformans. Cryptococcal Kcs1 converts IP6 to PP-IP5/IP7, but the kinase converting IP5 to IP6 is unknown. Deletion of a putative IP5 kinase-encoding gene (IPK1) alone (ipk1Δ), and in combination with KCS1 (ipk1Δkcs1Δ), profoundly reduced virulence in mice. However, deletion of KCS1 and IPK1 had a greater impact on virulence attenuation than that of IPK1 alone. ipk1Δkcs1Δ and kcs1Δ lung burdens were also lower than those of ipk1Δ. Unlike ipk1Δ, ipk1Δkcs1Δ and kcs1Δ failed to disseminate to the brain. IP profiling confirmed Ipk1 as the major IP5 kinase in C. neoformans: ipk1Δ produced no IP6 or PP-IP5/IP7 and, in contrast to ipk1Δkcs1Δ, accumulated IP5 and its pyrophosphorylated PP-IP4 derivative. Kcs1 is therefore a dual specificity (IP5 and IP6) kinase producing PP-IP4 and PP-IP5/IP7. All mutants were similarly attenuated in virulence phenotypes including laccase, urease and growth under oxidative/nitrosative stress. Alternative carbon source utilisation was also reduced significantly in all mutants except ipk1Δ, suggesting that PP-IP4 partially compensates for absent PP-IP5/IP7 in ipk1Δ grown under this condition. In conclusion, PP-IP5/IP7, not IP6, is essential for fungal virulence. PMID:27033523

  5. Lenz-Majewski mutations in PTDSS1 affect phosphatidylinositol 4-phosphate metabolism at ER-PM and ER-Golgi junctions

    PubMed Central

    Sohn, Mira; Ivanova, Pavlina; Brown, H. Alex; Varnai, Peter; Kim, Yeun Ju; Balla, Tamas

    2016-01-01

    Lenz-Majewski syndrome (LMS) is a rare disease characterized by complex craniofacial, dental, cutaneous, and limb abnormalities combined with intellectual disability. Mutations in the PTDSS1 gene coding one of the phosphatidylserine (PS) synthase enzymes, PSS1, were described as causative in LMS patients. Such mutations render PSS1 insensitive to feedback inhibition by PS levels. Here we show that expression of mutant PSS1 enzymes decreased phosphatidylinositol 4-phosphate (PI4P) levels both in the Golgi and the plasma membrane (PM) by activating the Sac1 phosphatase and altered PI4P cycling at the PM. Conversely, inhibitors of PI4KA, the enzyme that makes PI4P in the PM, blocked PS synthesis and reduced PS levels by 50% in normal cells. However, mutant PSS1 enzymes alleviated the PI4P dependence of PS synthesis. Oxysterol-binding protein–related protein 8, which was recently identified as a PI4P-PS exchanger between the ER and PM, showed PI4P-dependent membrane association that was significantly decreased by expression of PSS1 mutant enzymes. Our studies reveal that PS synthesis is tightly coupled to PI4P-dependent PS transport from the ER. Consequently, PSS1 mutations not only affect cellular PS levels and distribution but also lead to a more complex imbalance in lipid homeostasis by disturbing PI4P metabolism. PMID:27044099

  6. Rhinovirus uses a phosphatidylinositol 4-phosphate/cholesterol counter-current for the formation of replication compartments at the ER-Golgi interface.

    PubMed

    Roulin, Pascal S; Lötzerich, Mark; Torta, Federico; Tanner, Lukas B; van Kuppeveld, Frank J M; Wenk, Markus R; Greber, Urs F

    2014-11-12

    Similar to other positive-strand RNA viruses, rhinovirus, the causative agent of the common cold, replicates on a web of cytoplasmic membranes, orchestrated by host proteins and lipids. The host pathways that facilitate the formation and function of the replication membranes and complexes are poorly understood. We show that rhinovirus replication depends on host factors driving phosphatidylinositol 4-phosphate (PI4P)-cholesterol counter-currents at viral replication membranes. Depending on the virus type, replication required phosphatidylinositol 4-kinase class 3beta (PI4K3b), cholesteryl-esterase hormone-sensitive lipase (HSL) or oxysterol-binding protein (OSBP)-like 1, 2, 5, 9, or 11 associated with lipid droplets, endosomes, or Golgi. Replication invariably required OSBP1, which shuttles cholesterol and PI4P between ER and Golgi at membrane contact sites. Infection also required ER-associated PI4P phosphatase Sac1 and phosphatidylinositol (PI) transfer protein beta (PITPb) shunting PI between ER-Golgi. These data support a PI4P-cholesterol counter-flux model for rhinovirus replication.

  7. Combretastatin-A4 phosphate improves the distribution and antitumor efficacy of albumin-bound paclitaxel in W256 breast carcinoma model

    PubMed Central

    Gao, Meng; Zhang, Dongjian; Jin, Qiaomei; Jiang, Cuihua; Wang, Cong; Li, Jindian; Peng, Fei; Huang, Dejian; Zhang, Jian; Song, Shaoli

    2016-01-01

    Nanomedicine holds great promise for fighting against malignant tumors. However, tumor elevated interstitial fluid pressure (IFP) seriously hinders convective transvascular and interstitial transport of nanomedicines and thus damages its antitumor efficacy. In this study, combretastatin-A4 phosphate (CA4P) was utilized to reduce tumor IFP, and thereby to improve the intratumoral distribution and antitumor efficacy of nanoparticle albumin-bound paclitaxel (nab-paclitaxel). IFP was measured using the wick-in-needle method in tumors growing subcutaneously pretreatment and posttreatment with a single intravenous injection of CA4P. The tracing method of iodine 131 isotope was used for biodistribution analysis of nab-paclitaxel. Liquid chromatography coupled with tandem mass spectrometry was used to detect the intratumoral concentration of paclitaxel. Magnetic resonance imaging was applied to monitor tumor volume and ratios of necrosis. The tumor IFP continued to decline gradually over time following CA4P treatment, reaching approximately 31% of the pretreatment value by 1 h posttreatment. Biodistribution data indicated that both 131I-nab-paclitaxel and paclitaxel exhibited higher tumor uptake in CA4P + 131I-nab-paclitaxel group compared with I131-nab-paclitaxel group. Nab-paclitaxel combined with CA4Pshowed significant tumor growth inhibition and higher tumor necrosis ratio relative to PBS, CA4P and nab-paclitaxel group, respectively. In conclusion, CA4P improved the intratumoral distribution and antitumor efficacy of nab-paclitaxel in W256 tumor-bearing rats. PMID:27531898

  8. Influence of Ca2+ and Mg2+ on the turnover of the phosphomonoester group of phosphatidylinositol 4-phosphate in human erythrocyte membranes.

    PubMed Central

    Hegewald, H; Müller, E; Klinger, R; Wetzker, R; Frunder, H

    1987-01-01

    In isolated erythrocyte membranes, increasing the free Mg2+ concentration from 0.5 to 10 mM progressively activates the membrane-bound phosphatidylinositol (PtdIns) kinase and leads to the establishment of a new equilibrium with higher phosphatidylinositol 4-phosphate (PtdIns4P) and lower PtdIns concentrations. The steady-state turnover of the phosphomonoester group of PtdIns4P also increases at high Mg2+ concentrations, indicating a simultaneous activation of PtdIns4P phosphomonoesterase by Mg2+. Half-maximum inhibition of PtdIns kinase occurs at 10 microM free Ca2+ in the presence of physiological free Mg2+ concentrations. Increasing free Mg2+ concentrations overcome Ca2+ inhibition of PtdIns kinase. In the presence of Ca2+, calmodulin activates Ca2+-transporting ATPase 5-fold, but does not alter pool size and radiolabelling of PtdIns4P. In intact erythrocytes, adding EGTA or EGTA plus Mg2+ and the ionophore A23187 to the external medium does not exert significant effects on concentration and radiolabelling of polyphosphoinositides when compared with controls in the presence of 1.4 mM free Ca2+. PMID:2821996

  9. Analysis of the metabolic turnover of the individual phosphate groups of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. Validation of novel analytical techniques by using 32P-labelled lipids from erythrocytes.

    PubMed Central

    Hawkins, P T; Michell, R H; Kirk, C J

    1984-01-01

    We have developed methods that yield estimates of the 32P content of each of the individual phosphate groups of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, thus extending the information available from studies of the labelling of these lipids in intact cells or membrane preparations. The analyses are undertaken with the deacylated lipids. Assay of the 5-phosphate of phosphatidylinositol 4,5-bisphosphate is achieved by the use, under conditions of first-order kinetics, of a 5-phosphate-specific phosphomonoesterase present in isolated erythrocyte membranes [Downes, Mussat & Michell (1982) Biochem. J. 203, 169-177]. Assay of the 4-phosphate of phosphatidylinositol 4-phosphate and of the total monoester phosphate content (4-phosphate plus 5-phosphate) of phosphatidylinositol 4,5-bisphosphate employs alkaline phosphatase from bovine intestine. The radioactivity of the 1-phosphate is that remaining as organic phosphate after exhaustive alkaline phosphatase treatment. The methodology has been validated by using lipids from human erythrocytes: these contain no 32P in their 1-phosphate. These methods should be of substantial value in studies of the many cells that show rapid hormonal perturbations of phosphatidylinositol 4,5-bisphosphate metabolism. PMID:6326746

  10. Overexpression of a novel activator of PAK4, the CDK5 kinase-associated protein CDK5RAP3, promotes hepatocellular carcinoma metastasis.

    PubMed

    Mak, Grace Wing-Yan; Chan, Mandy Man-Lok; Leong, Veronica Yee-Law; Lee, Joyce Man-Fong; Yau, Tai-On; Ng, Irene Oi-Lin; Ching, Yick-Pang

    2011-04-15

    The CDK5 kinase regulatory subunit-associated protein 3 (CDK5RAP3 or C53/LZAP) regulates apoptosis induced by genotoxic stress. Although CDK5RAP3 has been implicated in cancer progression, its exact role in carcinogenesis is not well established. In this article, we report that CDK5RAP3 has an important prometastatic function in hepatocarcinogenesis. An examination of human hepatocellular carcinoma (HCC) samples revealed at least twofold overexpression of CDK5RAP3 transcripts in 58% (39/67) of HCC specimens when compared with corresponding nontumorous livers. CDK5RAP3 overexpression was associated with more aggressive biological behavior. In HCC cell lines, stable overexpression of CDK5RAP3 promoted, and small interfering RNA-mediated knockdown inhibited, tumorigenic activity and metastatic potential. We found that overexpression of CDK5RAP3 and p21-activated protein kinase 4 (PAK4) correlated in human HCCs, and that CDK5RAP3 was a novel binding partner of PAK4, and this binding enhanced PAK4 activity. siRNA-mediated knockdown of PAK4 in CDK5RAP3-expressing HCC cells reversed the enhanced cell invasiveness mediated by CDK5RAP3 overexpression, implying that PAK4 is essential for CDK5RAP3 function. Taken together, our findings reveal that CDK5RAP3 is widely overexpressed in HCC and that overexpression of CDK5RAP3 promotes HCC metastasis through PAK4 activation.

  11. Ste12/Fab1 phosphatidylinositol-3-phosphate 5-kinase is required for nitrogen-regulated mitotic commitment and cell size control

    PubMed Central

    Schauries, Marie; Kaczmarek, Adrian; Franz-Wachtel, Mirita; Du, Wei; Krug, Karsten; Maček, Boris; Petersen, Janni

    2017-01-01

    Tight coupling of cell growth and cell cycle progression enable cells to adjust their rate of division, and therefore size, to the demands of proliferation in varying nutritional environments. Nutrient stress promotes inhibition of Target Of Rapamycin Complex 1 (TORC1) activity. In fission yeast, reduced TORC1 activity advances mitotic onset and switches growth to a sustained proliferation at reduced cell size. A screen for mutants, that failed to advance mitosis upon nitrogen stress, identified a mutant in the PIKFYVE 1-phosphatidylinositol-3-phosphate 5-kinase fission yeast homolog Ste12. Ste12PIKFYVE deficient mutants were unable to advance the cell cycle to reduce cell size after a nitrogen downshift to poor nitrogen (proline) growth conditions. While it is well established that PI(3,5)P2 signalling is required for autophagy and that Ste12PIKFYVE mutants have enlarged vacuoles (yeast lysosomes), neither a block to autophagy or mutants that independently have enlarged vacuoles had any impact upon nitrogen control of mitotic commitment. The addition of rapamycin to Ste12PIKFYVE deficient mutants reduced cell size at division to suggest that Ste12PIKFYVE possibly functions upstream of TORC1. ste12 mutants display increased Torin1 (TOR inhibitor) sensitivity. However, no major impact on TORC1 or TORC2 activity was observed in the ste12 deficient mutants. In summary, Ste12PIKFYVE is required for nitrogen-stress mediated advancement of mitosis to reduce cell size at division. PMID:28273166

  12. Assessment of Tumor Response to the Vascular Disrupting Agents 5,6-Dimethylxanthenone-4-Acetic Acid or Combretastatin-A4-Phosphate by Intrinsic Susceptibility Magnetic Resonance Imaging

    SciTech Connect

    McPhail, Lesley D. Griffiths, John R. D.Phil.; Robinson, Simon P.

    2007-11-15

    Purpose: To investigate the use of the transverse magnetic resonance imaging (MRI) relaxation rate R{sub 2}* (s{sup -1}) as a biomarker of tumor vascular response to monitor vascular disrupting agent (VDA) therapy. Methods and Materials: Multigradient echo MRI was used to quantify R{sub 2}* in rat GH3 prolactinomas. R{sub 2}* is a sensitive index of deoxyhemoglobin in the blood and can therefore be used to give an index of tissue oxygenation. Tumor R{sub 2}* was measured before and up to 35 min after treatment, and 24 h after treatment with either 350 mg/kg 5,6-dimethylxanthenone-4-acetic acid (DMXAA) or 100 mg/kg combretastatin-A4-phosphate (CA4P). After acquisition of the MRI data, functional tumor blood vessels remaining after VDA treatment were quantified using fluorescence microscopy of the perfusion marker Hoechst 33342. Results: DMXAA induced a transient, significant (p < 0.05) increase in tumor R{sub 2}* 7 min after treatment, whereas CA4P induced no significant changes in tumor R{sub 2}* over the first 35 min. Twenty-four hours after treatment, some DMXAA-treated tumors demonstrated a decrease in R{sub 2}*, but overall, reduction in R{sub 2}* was not significant for this cohort. Tumors treated with CA4P showed a significant (p < 0.05) reduction in R{sub 2}* 24 h after treatment. The degree of Hoechst 33342 uptake was associated with the degree of R{sub 2}* reduction at 24 h for both agents. Conclusions: The reduction in tumor R{sub 2}* or deoxyhemoglobin levels 24 h after VDA treatment was a result of reduced blood volume caused by prolonged vascular collapse. Our results suggest that DMXAA was less effective than CA4P in this rat tumor model.

  13. In Vivo Near-Infrared Spectroscopy and Magnetic Resonance Imaging Monitoring of Tumor Response to Combretastatin A-4-Phosphate Correlated With Therapeutic Outcome

    SciTech Connect

    Zhao Dawen; Chang Chenghui; Kim, Jae G.; Liu Hanli; Mason, Ralph P.

    2011-06-01

    Purpose: To develop a combination treatment consisting of combretastatin A-4-phosphate (CA4P) with radiation based on tumor oxygenation status. Methods and Materials: In vivo near-infrared spectroscopy (NIRS) and diffusion-weighted (DW) magnetic resonance imaging (MRI) were applied to noninvasively monitor changes in tumor blood oxygenation and necrosis induced by CA4P (30 mg/kg) in rat mammary 13762NF adenocarcinoma, and the evidence was used to optimize combinations of CA4P and radiation treatment (a single dose of 5 Gy). Results: NIRS showed decreasing concentrations of tumor vascular oxyhemoglobin and total hemoglobin during the first 2 h after CA4P treatment, indicating significant reductions in tumor blood oxygenation and perfusion levels (p < 0.001). Twenty-four hours later, in response to oxygen inhalation, significant recovery was observed in tumor vascular and tissue oxygenation according to NIRS and pimonidazole staining results, respectively (p < 0.05). DW MRI revealed significantly increased water diffusion in tumors measured by apparent diffusion coefficient at 24 h (p < 0.05), suggesting that CA4P-induced central necrosis. In concordance with the observed tumor oxygen dynamics, we found that treatment efficacy depended on the timing of the combined therapy. The most significant delay in tumor growth was seen in the group of tumors treated with radiation while the rats breathed oxygen 24 h after CA4P administration. Conclusions: Noninvasive evaluation of tumor oxygen dynamics allowed us to rationally enhance the response of syngeneic rat breast tumors to combined treatment of CA4P with radiation.

  14. Chloroplast localization of methylerythritol 4-phosphate pathway enzymes and regulation of mitochondrial genes in ispD and ispE albino mutants in Arabidopsis.

    PubMed

    Hsieh, Ming-Hsiun; Chang, Chiung-Yun; Hsu, Shih-Jui; Chen, Ju-Jiun

    2008-04-01

    Plant isoprenoids are derived from two independent pathways, the cytosolic mevalonate pathway and the plastid methylerythritol 4-phosphate (MEP) pathway. We used green fluorescent fusion protein assays to demonstrate that the Arabidopsis MEP pathway enzymes are localized to the chloroplast. We have also characterized three Arabidopsis albino mutants, ispD-1, ispD-2 and ispE-1, which have T-DNA insertions in the IspD and IspE genes of the MEP pathway. Levels of photosynthetic pigments are almost undetectable in these albino mutants. Instead of thylakoids, the ispD and ispE mutant chloroplasts are filled with large vesicles. Impairments in chloroplast development and functions may signal changes in the expression of nuclear, chloroplast and mitochondrial genes. We used northern blot analysis to examine the expression of photosynthetic and respiratory genes in the ispD and ispE albino mutants. Steady-state mRNA levels of nucleus- and chloroplast-encoded photosynthetic genes are significantly decreased in the albino mutants. In contrast, transcript levels of nuclear and mitochondrial genes encoding subunits of the mitochondrial electron transport chain are increased or not affected in these mutants. Genomic Southern blot analysis revealed that the DNA amounts of mitochondrial genes are not enhanced in the ispD and ispE albino mutants. These results support the notion that the functional state of chloroplasts may affect the expression of nuclear and mitochondrial genes. The up-regulation of mitochondrial genes in the albino mutants is not caused by changes of mitochondrial DNA copy number in Arabidopsis.

  15. Type I γ Phosphatidylinositol Phosphate 5-Kinase i5 Controls the Ubiquitination and Degradation of the Tumor Suppressor Mitogen-inducible Gene 6.

    PubMed

    Sun, Ming; Cai, Jinyang; Anderson, Richard A; Sun, Yue

    2016-10-07

    Mitogen-inducible gene 6 (Mig6) is a tumor suppressor, and the disruption of Mig6 expression is associated with cancer development. Mig6 directly interacts with epidermal growth factor receptor (EGFR) to suppress the activation and downstream signaling of EGFR. Therefore, loss of Mig6 enhances EGFR-mediated signaling and promotes EGFR-dependent carcinogenesis. The molecular mechanism modulating Mig6 expression in cancer remains unclear. Here we demonstrate that type I γ phosphatidylinositol phosphate 5-kinase i5 (PIPKIγi5), an enzyme producing phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), stabilizes Mig6 expression. Knockdown of PIPKIγi5 leads to the loss of Mig6 expression, which dramatically enhances and prolongs EGFR-mediated cell signaling. Loss of PIPKIγi5 significantly promotes Mig6 protein degradation via proteasomes, but it does not affect the Mig6 mRNA level. PIPKIγi5 directly interacts with the E3 ubiquitin ligase neuronal precursor cell-expressed developmentally down-regulated 4-1 (NEDD4-1). The C-terminal domain of PIPKIγi5 and the WW1 and WW2 domains of NEDD4-1 are required for their interaction. The C2 domain of NEDD4-1 is required for its interaction with PtdIns(4,5)P2 By binding with NEDD4-1 and producing PtdIns(4,5)P2, PIPKIγi5 perturbs NEDD4-1-mediated Mig6 ubiquitination and the subsequent proteasomal degradation. Thus, loss of NEDD4-1 can rescue Mig6 expression in PIPKIγi5 knockdown cells. In this way, PIPKIγi5, NEDD4-1, and Mig6 form a novel molecular nexus that controls EGFR activation and downstream signaling.

  16. The 2-C-methylerythritol 4-phosphate pathway in melon is regulated by specialized isoforms for the first and last steps

    PubMed Central

    Saladié, Montserrat; Wright, Louwrance P.; Garcia-Mas, Jordi; Rodriguez-Concepcion, Manuel; Phillips, Michael A.

    2014-01-01

    The 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway provides the precursors for the biosynthesis of plastidial isoprenoids, which include the carotenoid pigments of many fruits. We have analysed the genes encoding the seven enzymes of the MEP pathway in melon (Cucumis melo L.) and determined that the first one, 1-deoxyxylulose 5-phosphate synthase (DXS), and the last one, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR), are represented in the genome as a small gene family and paralogous pair, respectively. In the case of DXS, three genes encode functional DXS activities which fall into previously established type I (CmDXS1) and II (CmDXS2a and CmDXS2b) categories, while a fourth DXS-like gene belonging to the type III group did not encode a protein with DXS activity. Their expression patterns and phylogenies suggest that CmDXS1 is functionally specialized for developmental and photosynthetic processes, while CmDXS2a and CmDXS2b are induced in flowers and ripening fruit of orange- (but not white-) fleshed varieties, coinciding with β-carotene accumulation. This is the first instance connecting type II DXS genes to specialized isoprenoid biosynthesis in the fruit of an agronomically important species. Two HDR paralogues were shown to encode functional enzymes, although only CmHDR1 was highly expressed in the tissues and developmental stages tested. Phylogenetic analysis showed that in cucurbits such as melon, these HDR paralogues probably arose through individual gene duplications in a common angiosperm ancestor, mimicking a prior division in gymnosperms, while other flowering plants, including apple, soy, canola, and poplar, acquired HDR duplicates recently as homoeologues through large-scale genome duplications. We report the influence of gene duplication history on the regulation of the MEP pathway in melon and the role of specialized MEP-pathway isoforms in providing precursors for β-carotene production in orange-fleshed melon varieties. PMID

  17. RalF-Mediated Activation of Arf6 Controls Rickettsia typhi Invasion by Co-Opting Phosphoinositol Metabolism

    PubMed Central

    Rennoll-Bankert, Kristen E.; Rahman, M. Sayeedur; Guillotte, Mark L.; Lehman, Stephanie S.; Beier-Sexton, Magda; Gillespie, Joseph J.

    2016-01-01

    Rickettsiae are obligate intracellular pathogens that induce their uptake into nonphagocytic cells; however, the events instigating this process are incompletely understood. Importantly, diverse Rickettsia species are predicted to utilize divergent mechanisms to colonize host cells, as nearly all adhesins and effectors involved in host cell entry are differentially encoded in diverse Rickettsia species. One particular effector, RalF, a Sec7 domain-containing protein that functions as a guanine nucleotide exchange factor of ADP-ribosylation factors (Arfs), is critical for Rickettsia typhi (typhus group rickettsiae) entry but pseudogenized or absent from spotted fever group rickettsiae. Secreted early during R. typhi infection, RalF localizes to the host plasma membrane and interacts with host ADP-ribosylation factor 6 (Arf6). Herein, we demonstrate that RalF activates Arf6, a process reliant on a conserved Glu within the RalF Sec7 domain. Furthermore, Arf6 is activated early during infection, with GTP-bound Arf6 localized to the R. typhi entry foci. The regulation of phosphatidylinositol 4-phosphate 5-kinase (PIP5K), which generates PI(4,5)P2, by activated Arf6 is instrumental for bacterial entry, corresponding to the requirement of PI(4,5)P2 for R. typhi entry. PI(3,4,5)P3 is then synthesized at the entry foci, followed by the accumulation of PI(3)P on the short-lived vacuole. Inhibition of phosphoinositide 3-kinases, responsible for the synthesis of PI(3,4,5)P3 and PI(3)P, negatively affects R. typhi infection. Collectively, these results identify RalF as the first bacterial effector to directly activate Arf6, a process that initiates alterations in phosphoinositol metabolism critical for a lineage-specific Rickettsia entry mechanism. PMID:27698019

  18. Influence of temperature and frequency on ionic conductivity of Li{sub 3}PO{sub 4}–Pb{sub 3}(PO{sub 4}){sub 2}–BiPO{sub 4} phosphate glasses

    SciTech Connect

    El Moudane, M.; El Maniani, M.; Sabbar, A.; Ghanimi, A.; Tabyaoui, M.; Bellaouchou, A.; Guenbour, A.

    2015-12-15

    Highlights: • Results of ionic conductivities of Li{sub 3}PO{sub 4}–Pb{sub 3}(PO{sub 4}){sub 2}–BiPO{sub 4} phosphate glasses. • Determination of glass transition temperature using DSC method. • Study of temperature and frequency on ionic conductivity of Li{sub 3}PO{sub 4}–Pb{sub 3}(PO{sub 4}){sub 2}–BiPO{sub 4} phosphate glasses. - Abstract: Lithium–Lead–Bismuth phosphates glasses having, a composition 30Li{sub 3}PO{sub 4}–(70 − x)Pb{sub 3}(PO{sub 4}){sub 2}–xBiPO{sub 4} (45 ≤ x ≤ 60 mol%) were prepared by using the melt quenching method 1000 °C. The thermal stability of theses glasses increases with the substitution of Bi{sub 2}O{sub 3} with PbO. The ionic conductivity of all compositions have been measured over a wide temperature (200–500 °C) and frequency range (1–106 Hz). The ionic conductivity data below and above T{sub g} follows Arrhenius and Vogel–Tamman–Fulcher (VTF) relationship, respectively. The activation energies are estimated and discussed. The dependence in frequency of AC conductivity is found to obey Jonscher’s relation.

  19. Escherichia coli engineered to synthesize isopentenyl diphosphate and dimethylallyl diphosphate from mevalonate: a novel system for the genetic analysis of the 2-C-methyl-d-erythritol 4-phosphate pathway for isoprenoid biosynthesis.

    PubMed Central

    Campos, N; Rodríguez-Concepción, M; Sauret-Güeto, S; Gallego, F; Lois, L M; Boronat, A

    2001-01-01

    Isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) constitute the basic building block of isoprenoids, a family of compounds that is extraordinarily diverse in structure and function. IPP and DMAPP can be synthesized by two independent pathways: the mevalonate pathway and the recently discovered 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. Although the MEP pathway is essential in most eubacteria, algae and plants and has enormous biotechnological interest, only some of its steps have been determined. We devised a system suitable for the genetic analysis of the MEP pathway in Escherichia coli. A synthetic operon coding for yeast 5-diphosphomevalonate decarboxylase, human 5-phosphomevalonate kinase, yeast mevalonate kinase and E. coli isopentenyl diphosphate isomerase was incorporated in the chromosome of this bacterium. The expression of this operon allowed the synthesis of IPP and DMAPP from mevalonate added exogenously and complementation of lethal mutants of the MEP pathway. We used this system to show that the ygbP, ychB and ygbB genes are essential in E. coli and that the steps catalysed by the products of these genes belong to the trunk line of the MEP pathway. PMID:11115399

  20. Deuterium-labelled isotopomers of 2-C-methyl-D-erythritol as tools for the elucidation of the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid biosynthesis.

    PubMed Central

    Charon, L; Hoeffler, J F; Pale-Grosdemange, C; Lois, L M; Campos, N; Boronat, A; Rohmer, M

    2000-01-01

    Escherichia coli synthesizes its isoprenoids via the mevalonate-independent 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. The MC4100dxs::CAT strain, defective in deoxyxylulose-5-phosphate synthase, which is the first enzyme in this metabolic route, exclusively synthesizes its isoprenoids from exogenous 2-C-methyl-D-erythritol (ME) added to the culture medium. The fate of the hydrogen atoms in the MEP pathway was followed by the incorporation of [1,1-(2)H(2)]ME and [3,5,5,5-(2)H(4)]ME. The two C-1 hydrogen atoms of ME were found without any loss in the prenyl chain of menaquinone and/or ubiquinone on the carbon atoms derived from C-4 of isopentenyl diphosphate (IPP) and on the E-methyl group of dimethylallyl diphosphate (DMAPP), the C-5 hydrogen atoms on the methyl groups derived from IPP C-5 methyl group and the Z-methyl group of DMAPP. This showed that no changes in the oxidation state of these carbon atoms occurred in the reaction sequence between MEP and IPP. Furthermore, no deuterium scrambling was observed between the carbon atoms derived from C-4 and C-5 of IPP or DMAPP, suggesting a completely stereoselective IPP isomerase or no significant activity of this enzyme. The C-3 deuterium atom of [3,5,5,5-(2)H(4)]ME was preserved only in the DMAPP starter unit and was completely missing from all those derived from IPP. This finding, aided by the non-essential role of the IPP isomerase gene, suggests the presence in E. coli of two different routes towards IPP and DMAPP, starting from a common intermediate derived from MEP. PMID:10698701

  1. A High and Phosphatidylinositol-4-phosphate (PI4P)-dependent ATPase Activity for the Drs2p/Cdc50p Flippase after Removal of its N- and C-terminal Extensions.

    PubMed

    Azouaoui, Hassina; Montigny, Cédric; Dieudonné, Thibaud; Champeil, Philippe; Jacquot, Aurore; Vázquez-Ibar, José Luis; Le Maréchal, Pierre; Ulstrup, Jakob; Ash, Miriam-Rose; Lyons, Joseph A; Nissen, Poul; Lenoir, Guillaume

    2017-03-16

    P4-ATPases, also known as phospholipid flippases, are responsible for creating and maintaining transbilayer lipid asymmetry in eukaryotic cell membranes. Here, we use limited proteolysis to investigate the role of the N- and C-termini in ATP hydrolysis and auto-inhibition of the yeast flippase Drs2p/Cdc50p. We show that limited proteolysis of the detergent-solubilized and purified yeast flippase may result in more than one order of magnitude increase of its ATPase activity, which remains dependent on phosphatidylinositol-4-phosphate (PI4P), a regulator of this lipid flippase, and specific to a phosphatidylserine substrate. Using thrombin as the protease, Cdc50p remains intact and in complex with Drs2p, which is cleaved at two positions, namely after R104 and after R1290, resulting in a homogenous sample lacking 104 and 65 residues from its N- and C-termini, respectively. Removal of the 1291-1302 region of the C-terminal extension is critical for relieving the auto-inhibition of full-length Drs2p, while the 1-104 N-terminal residues have an additional but more modest significance for activity. The present results therefore reveal that trimming off appropriate regions of the terminal extensions of Drs2p can greatly increase its ATPase activity in the presence of PI4P, and demonstrate that relief of such auto-inhibition remains compatible with subsequent regulation by PI4P. These experiments suggest that activation of the Drs2p/Cdc50p flippase follows a multi-step mechanism, with preliminary release of a number of constraints, possibly through the binding of regulatory proteins in the trans-Golgi network, followed by full activation by PI4P.

  2. A High-Yield Co-Expression System for the Purification of an Intact Drs2p-Cdc50p Lipid Flippase Complex, Critically Dependent on and Stabilized by Phosphatidylinositol-4-Phosphate

    PubMed Central

    Azouaoui, Hassina; Montigny, Cédric; Ash, Miriam-Rose; Fijalkowski, Frank; Jacquot, Aurore; Grønberg, Christina; López-Marqués, Rosa L.; Palmgren, Michael G.; Garrigos, Manuel; le Maire, Marc; Decottignies, Paulette; Gourdon, Pontus; Nissen, Poul; Champeil, Philippe; Lenoir, Guillaume

    2014-01-01

    P-type ATPases from the P4 subfamily (P4-ATPases) are energy-dependent transporters, which are thought to establish lipid asymmetry in eukaryotic cell membranes. Together with their Cdc50 accessory subunits, P4-ATPases couple ATP hydrolysis to lipid transport from the exoplasmic to the cytoplasmic leaflet of plasma membranes, late Golgi membranes, and endosomes. To gain insights into the structure and function of these important membrane pumps, robust protocols for expression and purification are required. In this report, we present a procedure for high-yield co-expression of a yeast flippase, the Drs2p-Cdc50p complex. After recovery of yeast membranes expressing both proteins, efficient purification was achieved in a single step by affinity chromatography on streptavidin beads, yielding ∼1–2 mg purified Drs2p-Cdc50p complex per liter of culture. Importantly, the procedure enabled us to recover a fraction that mainly contained a 1∶1 complex, which was assessed by size-exclusion chromatography and mass spectrometry. The functional properties of the purified complex were examined, including the dependence of its catalytic cycle on specific lipids. The dephosphorylation rate was stimulated in the simultaneous presence of the transported substrate, phosphatidylserine (PS), and the regulatory lipid phosphatidylinositol-4-phosphate (PI4P), a phosphoinositide that plays critical roles in membrane trafficking events from the trans-Golgi network (TGN). Likewise, overall ATP hydrolysis by the complex was critically dependent on the simultaneous presence of PI4P and PS. We also identified a prominent role for PI4P in stabilization of the Drs2p-Cdc50p complex towards temperature- or C12E8-induced irreversible inactivation. These results indicate that the Drs2p-Cdc50p complex remains functional after affinity purification and that PI4P as a cofactor tightly controls its stability and catalytic activity. This work offers appealing perspectives for detailed structural and

  3. Regulation of the Na(+)-coupled glutamate transporter EAAT3 by PIKfyve.

    PubMed

    Klaus, Fabian; Gehring, Eva-Maria; Zürn, Agathe; Laufer, Joerg; Lindner, Ricco; Strutz-Seebohm, Nathalie; Tavaré, Jeremy M; Rothstein, Jeffrey D; Boehmer, Christoph; Palmada, Monica; Gruner, Ivonne; Lang, Undine E; Seebohm, Guiscard; Lang, Florian

    2009-01-01

    The Na(+), glutamate cotransporter EAAT3 is expressed in a wide variety of tissues. It accomplishes transepithelial transport and the cellular uptake of acidic amino acids. Regulation of EAAT3 activity involves a signaling cascade including the phosphatidylinositol-3 (PI3)-kinase, the phosphoinositide dependent kinase PDK1, and the serum and glucocorticoid inducible kinase SGK1. Targets of SGK1include the mammalian phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3). The present experiments explored whether PIKfyve participates in the regulation of EAAT3 activity. To this end,EAAT3 was expressed in Xenopus oocytes with or without SGK1 and/or PIKfyve and glutamate-induced current (I(glu)) determined by dual electrode voltage clamp. In Xenopus oocytes expressing EAAT3 but not in water injected oocytes glutamate induced an inwardly directed I(glu). Coexpression of either, SGK1 orPIKfyve, significantly enhanced I(glu) in EAAT3 expressing oocytes. The increased I(glu) was paralleled by increased EAAT3 protein abundance in the oocyte cell membrane. I(glu) and EAAT3 protein abundance were significantly larger in oocytes coexpressing EAAT3, SGK1 and PIKfyve than in oocytes expressingEAAT3 and either, SGK1 or PIKfyve, alone. Coexpression of the inactive SGK1 mutant (K127N)SGK1 did not significantly alter I(glu) in EAAT3 expressing oocytes and completely reversed the stimulating effect ofPIKfyve coexpression on I(glu). The stimulating effect of PIKfyve on I(glu) was abolished by replacement of the serine by alanine in the SGK consensus sequence ((S318A)PIKfyve). Moreover, additional coexpression of(S318A)PIKfyve significantly blunted I(glu) in Xenopus oocytes coexpressing SGK1 and EAAT3. The observations demonstrate that PIKfyve participates in EAAT3 regulation likely downstream of SGK1.

  4. Early-onset epileptic encephalopathy caused by a reduced sensitivity of Kv7.2 potassium channels to phosphatidylinositol 4,5-bisphosphate

    PubMed Central

    Soldovieri, Maria Virginia; Ambrosino, Paolo; Mosca, Ilaria; De Maria, Michela; Moretto, Edoardo; Miceli, Francesco; Alaimo, Alessandro; Iraci, Nunzio; Manocchio, Laura; Medoro, Alessandro; Passafaro, Maria; Taglialatela, Maurizio

    2016-01-01

    Kv7.2 and Kv7.3 subunits underlie the M-current, a neuronal K+ current characterized by an absolute functional requirement for phosphatidylinositol 4,5-bisphosphate (PIP2). Kv7.2 gene mutations cause early-onset neonatal seizures with heterogeneous clinical outcomes, ranging from self-limiting benign familial neonatal seizures to severe early-onset epileptic encephalopathy (Kv7.2-EE). In this study, the biochemical and functional consequences prompted by a recurrent variant (R325G) found independently in four individuals with severe forms of neonatal-onset EE have been investigated. Upon heterologous expression, homomeric Kv7.2 R325G channels were non-functional, despite biotin-capture in Western blots revealed normal plasma membrane subunit expression. Mutant subunits exerted dominant-negative effects when incorporated into heteromeric channels with Kv7.2 and/or Kv7.3 subunits. Increasing cellular PIP2 levels by co-expression of type 1γ PI(4)P5-kinase (PIP5K) partially recovered homomeric Kv7.2 R325G channel function. Currents carried by heteromeric channels incorporating Kv7.2 R325G subunits were more readily inhibited than wild-type channels upon activation of a voltage-sensitive phosphatase (VSP), and recovered more slowly upon VSP switch-off. These results reveal for the first time that a mutation-induced decrease in current sensitivity to PIP2 is the primary molecular defect responsible for Kv7.2-EE in individuals carrying the R325G variant, further expanding the range of pathogenetic mechanisms exploitable for personalized treatment of Kv7.2-related epilepsies. PMID:27905566

  5. Tamoxifen inhibits CDK5 kinase activity by interacting with p35/p25 and modulates the pattern of tau phosphorylation.

    PubMed

    Corbel, Caroline; Zhang, Bing; Le Parc, Annabelle; Baratte, Blandine; Colas, Pierre; Couturier, Cyril; Kosik, Kenneth S; Landrieu, Isabelle; Le Tilly, Véronique; Bach, Stéphane

    2015-04-23

    Cyclin-dependent kinase 5 (CDK5) is a multifunctional enzyme that plays numerous roles, notably in brain development. CDK5 is activated through its association with the activators, p35 and p39, rather than by cyclins. Proteolytic procession of the N-terminal part of its activators has been linked to Alzheimer's disease and various other neuropathies. The interaction with the proteolytic product p25 prolongs CDK5 activation and modifies the substrate specificity. In order to discover small-molecule inhibitors of the interaction between CDK5 and p25, we have used a bioluminescence resonance energy transfer (BRET)-based screening assay. Among the 1,760 compounds screened, the generic drug tamoxifen has been identified. The inhibition of the CDK5 activity by tamoxifen was notably validated by monitoring the phosphorylation state of tau protein. The study of the molecular mechanism of inhibition indicates that tamoxifen interacts with p25 to block the CDK5/p25 interaction and pave the way for new treatments of tauopathies.

  6. Activated type I TGFbeta receptor (Alk5) kinase confers enhancedsurvival to mammary epithelial cells and accelerates mammary tumorprogression

    SciTech Connect

    Muraoka-Cook, Rebecca S.; Shin, Incheol; Yi, Jae Youn; Easterly,Evangeline; Barcellos-Hoff, Mary Helen; Yingling, Jonathan M.; Zent, Roy; Arteaga, Carlos L.

    2005-01-02

    The transforming growth factor-betas (TGF{beta}s) are members of a large superfamily of pleiotropic cytokines that also includes the activins and the bone morphogenetic proteins (BMPs). Members of the TGF{beta} family regulate complex physiological processes such cell proliferation, differentiation, adhesion, cell-cell and cell-matrix interactions, motility, and cell death, among others (Massague, 1998). Dysregulation of TGF{beta} signaling contributes to several pathological processes including cancer, fibrosis, and auto-immune disorders (Massague et al., 2000). The TGF{beta}s elicit their biological effects by binding to type II and type I transmembrane receptor serine-threonine kinases (T{beta}RII and T{beta}RI) which, in turn, phosphorylated Smad 2 and Smad 3. Phosphorylated Smad 2/3 associate with Smad 4 and, as a heteromeric complex, translocate to the nucleus where they regulate gene transcription. The inhibitory Smad7 down regulates TGF{beta} signaling by binding to activated T{beta}RI and interfering with its ability to phosphorylate Smad 2/3 (Derynck and Zhang, 2003; Shi and Massague, 2003). Signaling is also regulated by Smad proteolysis. TGF{beta} receptor-mediated activation results in multi-ubiquitination of Smad 2 in the nucleus and subsequent degradation of Smad 2 by the proteasome (Lo and Massague, 1999). Activation of TGF{beta} receptors also induces mobilization of a Smad 7-Smurf complex from the nucleus to the cytoplasm; this complex recognizes the activated receptors and mediates their ubiquitination and internalization via caveolin-rich vesicles, leading to termination of TGF{beta} signaling (Di Guglielmo et al., 2003). Other signal transducers/pathways have been implicated in TGF{beta} actions. These include the extracellular signal-regulated kinase (Erk), c-Jun N-terminal kinase (Jnk), p38 mitogen-activated protein kinase (MAPK), protein phosphatase PP2A, phosphatidylinositol-3 kinase (PI3K), and the family of Rho GTPases [reviewed in (Derynck and Zhang, 2003)]. Although signaling by Smads has been shown to be causally associated with the anti-proliferative effect of TGF{beta} (Datto et al., 1999; Liu et al., 1997), the role of non-Smad effectors on mediating the cellular effects of TGF{beta} is less well characterized.

  7. Ca2+ Influx through Store-operated Calcium Channels Replenishes the Functional Phosphatidylinositol 4,5-Bisphosphate Pool Used by Cysteinyl Leukotriene Type I Receptors.

    PubMed

    Alswied, Abdullah; Parekh, Anant B

    2015-12-04

    Oscillations in cytoplasmic Ca(2+) concentration are a universal mode of signaling following physiological levels of stimulation with agonists that engage the phospholipase C pathway. Sustained cytoplasmic Ca(2+) oscillations require replenishment of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2), the source of the Ca(2+)-releasing second messenger inositol trisphosphate. Here we show that cytoplasmic Ca(2+) oscillations induced by cysteinyl leukotriene type I receptor activation run down when cells are pretreated with Li(+), an inhibitor of inositol monophosphatases that prevents PIP2 resynthesis. In Li(+)-treated cells, cytoplasmic Ca(2+) signals evoked by an agonist were rescued by addition of exogenous inositol or phosphatidylinositol 4-phosphate (PI4P). Knockdown of the phosphatidylinositol 4-phosphate 5 (PIP5) kinases α and γ resulted in rapid loss of the intracellular Ca(2+) oscillations and also prevented rescue by PI4P. Knockdown of talin1, a protein that helps regulate PIP5 kinases, accelerated rundown of cytoplasmic Ca(2+) oscillations, and these could not be rescued by inositol or PI4P. In Li(+)-treated cells, recovery of the cytoplasmic Ca(2+) oscillations in the presence of inositol or PI4P was suppressed when Ca(2+) influx through store-operated Ca(2+) channels was inhibited. After rundown of the Ca(2+) signals following leukotriene receptor activation, stimulation of P2Y receptors evoked prominent inositol trisphosphate-dependent Ca(2+) release. Therefore, leukotriene and P2Y receptors utilize distinct membrane PIP2 pools. Our findings show that store-operated Ca(2+) entry is needed to sustain cytoplasmic Ca(2+) signaling following leukotriene receptor activation both by refilling the Ca(2+) stores and by helping to replenish the PIP2 pool accessible to leukotriene receptors, ostensibly through control of PIP5 kinase activity.

  8. siRNA Screen Identifies Trafficking Host Factors that Modulate Alphavirus Infection

    DTIC Science & Technology

    2016-05-20

    chloramphenicol acetyltransferase ( CAT ), wild-type Rac1, or variants thereof. Cells were fixed 170 18 h (VEEV) or 24 h (RVFV) after virus inoculation and stained... CAT , wild-type Rac1, or 174 Rac1 K186E. Cells were infected and stained as in (F). 175 176 Rac1 GTPase Function and Rac1:PIP5K1-α Complex...179 established tetracycline-inducible 293 Flp-In T-REx cell lines that express chloramphenicol 180 acetyltransferase ( CAT , used as a control), wild

  9. Transient and sustained increases in inositol 1,4,5-trisphosphate precede the differential growth response in gravistimulated maize pulvini

    NASA Technical Reports Server (NTRS)

    Perera, I. Y.; Heilmann, I.; Boss, W. F.; Davies, E. (Principal Investigator)

    1999-01-01

    The internodal maize pulvinus responds to gravistimulation with differential cell elongation on the lower side. As the site of both graviperception and response, the pulvinus is an ideal system to study how organisms sense changes in orientation. We observed a transient 5-fold increase in inositol 1,4,5-trisphosphate (IP3) within 10 s of gravistimulation in the lower half of the pulvinus, indicating that the positional change was sensed immediately. Over the first 30 min, rapid IP3 fluctuations were observed between the upper and lower halves. Maize plants require a presentation time of between 2 and 4 h before the cells on the lower side of the pulvinus are committed to elongation. After 2 h of gravistimulation, the lower half consistently had higher IP3, and IP3 levels on the lower side continued to increase up to approximately 5-fold over basal levels before visible growth. As bending became visible after 8-10 h, IP3 levels returned to basal values. Additionally, phosphatidylinositol 4-phosphate 5-kinase activity in the lower pulvinus half increased transiently within 10 min of gravistimulation, suggesting that the increased IP3 production was accompanied by an up-regulation of phosphatidylinositol 4, 5-bisphosphate biosynthesis. Neither IP3 levels nor phosphatidylinositol 4-phosphate 5-kinase activity changed in pulvini halves from vertical control plants. Our data indicate the involvement of IP3 and inositol phospholipids in both short- and long-term responses to gravistimulation. As a diffusible second messenger, IP3 provides a mechanism to transmit and amplify the signal from the perceiving to the responding cells in the pulvinus, coordinating a synchronized growth response.

  10. siRNA Screen Identifies Trafficking Host Factors that Modulate Alphavirus Infection

    PubMed Central

    Radoshitzky, Sheli R.; Pegoraro, Gianluca; Chī, Xiǎolì; Dǒng, Lián; Chiang, Chih-Yuan; Jozwick, Lucas; Clester, Jeremiah C.; Cooper, Christopher L.; Courier, Duane; Langan, David P.; Underwood, Knashka; Kuehl, Kathleen A.; Sun, Mei G.; Caì, Yíngyún; Yú, Shuǐqìng; Burk, Robin; Zamani, Rouzbeh; Kota, Krishna; Kuhn, Jens H.; Bavari, Sina

    2016-01-01

    Little is known about the repertoire of cellular factors involved in the replication of pathogenic alphaviruses. To uncover molecular regulators of alphavirus infection, and to identify candidate drug targets, we performed a high-content imaging-based siRNA screen. We revealed an actin-remodeling pathway involving Rac1, PIP5K1- α, and Arp3, as essential for infection by pathogenic alphaviruses. Infection causes cellular actin rearrangements into large bundles of actin filaments termed actin foci. Actin foci are generated late in infection concomitantly with alphavirus envelope (E2) expression and are dependent on the activities of Rac1 and Arp3. E2 associates with actin in alphavirus-infected cells and co-localizes with Rac1–PIP5K1-α along actin filaments in the context of actin foci. Finally, Rac1, Arp3, and actin polymerization inhibitors interfere with E2 trafficking from the trans-Golgi network to the cell surface, suggesting a plausible model in which transport of E2 to the cell surface is mediated via Rac1- and Arp3-dependent actin remodeling. PMID:27031835

  11. siRNA Screen Identifies Trafficking Host Factors that Modulate Alphavirus Infection.

    PubMed

    Radoshitzky, Sheli R; Pegoraro, Gianluca; Chī, Xi Olì; D Ng, Lián; Chiang, Chih-Yuan; Jozwick, Lucas; Clester, Jeremiah C; Cooper, Christopher L; Courier, Duane; Langan, David P; Underwood, Knashka; Kuehl, Kathleen A; Sun, Mei G; Caì, Yíngyún; Yú, Shu Qìng; Burk, Robin; Zamani, Rouzbeh; Kota, Krishna; Kuhn, Jens H; Bavari, Sina

    2016-03-01

    Little is known about the repertoire of cellular factors involved in the replication of pathogenic alphaviruses. To uncover molecular regulators of alphavirus infection, and to identify candidate drug targets, we performed a high-content imaging-based siRNA screen. We revealed an actin-remodeling pathway involving Rac1, PIP5K1- α, and Arp3, as essential for infection by pathogenic alphaviruses. Infection causes cellular actin rearrangements into large bundles of actin filaments termed actin foci. Actin foci are generated late in infection concomitantly with alphavirus envelope (E2) expression and are dependent on the activities of Rac1 and Arp3. E2 associates with actin in alphavirus-infected cells and co-localizes with Rac1-PIP5K1-α along actin filaments in the context of actin foci. Finally, Rac1, Arp3, and actin polymerization inhibitors interfere with E2 trafficking from the trans-Golgi network to the cell surface, suggesting a plausible model in which transport of E2 to the cell surface is mediated via Rac1- and Arp3-dependent actin remodeling.

  12. A PLCβ/PI3Kγ-GSK3 signaling pathway regulates cofilin phosphatase slingshot2 and neutrophil polarization and chemotaxis

    PubMed Central

    Tang, Wenwen; Zhang, Yong; Xu, Wenwen; Harden, T. Kendall; Sondek, John; Sun, Le; Li, Lin

    2011-01-01

    SUMMARY Neutrophils, in response to a chemoattractant gradient, undergo dynamic F actin remodeling, a process important for their directional migration or chemotaxis. However, signaling mechanisms for chemoattractants to regulate the process are incompletely understood. Here, we characterized chemoattractant-activated signaling mechanisms that regulate cofilin dephosphorylation and actin cytoskeleton reorganization and are critical for neutrophil polarization and chemotaxis. In neutrophils, chemoattractants induced phosphorylation and inhibition of GSK3 via both PLCβ-PKC and PI3Kγ-AKT pathways, leading to the attenuation of GSK3-mediated phosphorylation and inhibition of the cofilin phosphatase slingshot2 and an increase in dephosphorylated, active cofilin. The relative contribution of this GSK3-mediated pathway to neutrophil chemotaxis regulation depended on neutrophil polarity preset by integrin-induced polarization of PIP5K1C. Therefore, our study characterizes a signaling mechanism for chemoattractant-induced actin cytoskeleton remodeling and elucidates its context-dependent role in regulating neutrophil polarization and chemotaxis. PMID:22172670

  13. Effect of Ovarian Hormones on Genes Promoting Dendritic Spines In Laser Captured Serotonin Neurons From Macaques

    PubMed Central

    Bethea, Cynthia L.; Reddy, Arubala P.

    2009-01-01

    Dendritic spines are the elementary structural units of neuronal plasticity and the cascades that promote dendrite spine remodeling center on Rho GTPases and downstream effectors of actin dynamics. In a model of hormone replacement therapy (HT), we sought the effect of estradiol (E) and progesterone (P) on gene expression in these cascades in laser captured serotonin neurons from rhesus macaques with cDNA array analysis. Spayed rhesus macaques were treated with either placebo, E or E+P via Silastic implant for 1 month prior to euthanasia after which the midbrain was obtained, sectioned and immunostained for TPH. TPH-positive neurons were laser captured using an Arcturus Laser Dissection Microscope (PixCell II). RNA from laser captured serotonin neurons (n=2 animals/treatment) was hybridized to Rhesus Affymetrix GeneChips. There was a significant change in 744 probe sets (ANOVA, p < 0.05), but 10,493 probe sets exhibited a 2-fold or greater change. Pivotal changes in pathways leading to dendrite spine proliferation and transformation included 2-fold or greater increases in expression of the Rho GTPases called CDC42, Rac1 and RhoA. In addition, 2-fold or greater increases occurred in downstream effectors of actin dynamics including PAK1, ROCK, PIP5K, IRSp53, WASP, WAVE, MLC, cofilin, gelsolin, profilin and 3 subunits of ARP2/3. Finally, 2-fold or greater decreases occurred in CRIPAK, LIMK2 and MLCK. The regulation of RhoA, Rac1, CDC42, ROCK, PIP5k, IRSp53, WASP, WAVE, LIMK2, CRIPAK1, MLCK, ARP2/3 subunit 3, gelsolin, profilin and cofilin was confirmed with nested qRT-PCR on laser captured RNA (n=3 animals/treatment). The data indicate that ovarian steroids target gene expression of the Rho GTPases and pivotal downstream proteins that in turn, would promote dendritic spine proliferation and stabilization on serotonin neurons of the dorsal raphe nucleus. PMID:19687787

  14. The essential phosphoinositide kinase MSS-4 is required for polar hyphal morphogenesis, localizing to sites of growth and cell fusion in Neurospora crassa.

    PubMed

    Mähs, Anette; Ischebeck, Till; Heilig, Yvonne; Stenzel, Irene; Hempel, Franziska; Seiler, Stephan; Heilmann, Ingo

    2012-01-01

    Fungal hyphae and plant pollen tubes are among the most highly polarized cells known and pose extraordinary requirements on their cell polarity machinery. Cellular morphogenesis is driven through the phospholipid-dependent organization at the apical plasma membrane. We characterized the contribution of phosphoinositides (PIs) in hyphal growth of the filamentous ascomycete Neurospora crassa. MSS-4 is an essential gene and its deletion resulted in spherically growing cells that ultimately lyse. Two conditional mss-4-mutants exhibited altered hyphal morphology and aberrant branching at restrictive conditions that were complemented by expression of wild type MSS-4. Recombinant MSS-4 was characterized as a phosphatidylinositolmonophosphate-kinase phosphorylating phosphatidylinositol 4-phosphate (PtdIns4P) to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). PtdIns3P was also used as a substrate. Sequencing of two conditional mss-4 alleles identified a single substitution of a highly conserved Y750 to N. The biochemical characterization of recombinant protein variants revealed Y750 as critical for PI4P 5-kinase activity of MSS-4 and of plant PI4P 5-kinases. The conditional growth defects of mss-4 mutants were caused by severely reduced activity of MSS-4(Y750N), enabling the formation of only trace amounts of PtdIns(4,5)P(2). In N. crassa hyphae, PtdIns(4,5)P(2) localized predominantly in the plasma membrane of hyphae and along septa. Fluorescence-tagged MSS-4 formed a subapical collar at hyphal tips, localized to constricting septa and accumulated at contact points of fusing N. crassa germlings, indicating MSS-4 is responsible for the formation of relevant pools of PtdIns(4,5)P(2) that control polar and directional growth and septation. N. crassa MSS-4 differs from yeast, plant and mammalian PI4P 5-kinases by containing additional protein domains. The N-terminal domain of N. crassa MSS-4 was required for correct membrane association. The data presented for N

  15. Phosphatidylinositol phosphate kinase PIPKIγ and phosphatase INPP5E coordinate initiation of ciliogenesis.

    PubMed

    Xu, Qingwen; Zhang, Yuxia; Wei, Qing; Huang, Yan; Hu, Jinghua; Ling, Kun

    2016-02-26

    Defective primary cilia are causative to a wide spectrum of human genetic disorders, termed ciliopathies. Although the regulation of ciliogenesis is intensively studied, how it is initiated remains unclear. Here we show that type Iγ phosphatidylinositol 4-phosphate (PtdIns(4)P) 5-kinase (PIPKIγ) and inositol polyphosphate-5-phosphatase E (INPP5E), a Joubert syndrome protein, localize to the centrosome and coordinate the initiation of ciliogenesis. PIPKIγ counteracts INPP5E in regulating tau-tubulin kinase-2 (TTBK2) recruitment to the basal body, which promotes the removal of microtubule capping protein CP110 and the subsequent axoneme elongation. Interestingly, INPP5E and its product--PtdIns(4)P--accumulate at the centrosome/basal body in non-ciliated, but not ciliated, cells. PtdIns(4)P binding to TTBK2 and the distal appendage protein CEP164 compromises the TTBK2-CEP164 interaction and inhibits the recruitment of TTBK2. Our results reveal that PtdIns(4)P homoeostasis, coordinated by PIPKIγ and INPP5E at the centrosome/ciliary base, is vital for ciliogenesis by regulating the CEP164-dependent recruitment of TTBK2.

  16. RdgBα reciprocally transfers PA and PI at ER-PM contact sites to maintain PI(4,5)P2 homoeostasis during phospholipase C signalling in Drosophila photoreceptors.

    PubMed

    Cockcroft, Shamshad; Garner, Kathryn; Yadav, Shweta; Gomez-Espinoza, Evelyn; Raghu, Padinjat

    2016-02-01

    Phosphatidylinositol (PI) is the precursor lipid for the synthesis of PI 4,5-bisphosphate [PI(4,5)P2] at the plasma membrane (PM) and is sequentially phosphorylated by the lipid kinases, PI 4-kinase and phosphatidylinositol 4-phosphate (PI4P)-5-kinase. Receptor-mediated hydrolysis of PI(4,5)P2 takes place at the PM but PI resynthesis occurs at the endoplasmic reticulum (ER). Thus PI(4,5)P2 resynthesis requires the reciprocal transport of two key intermediates, phosphatidic acid (PA) and PI between the ER and the PM. PI transfer proteins (PITPs), defined by the presence of the PITP domain, can facilitate lipid transfer between membranes; the PITP domain comprises a hydrophobic cavity with dual specificity but accommodates a single phospholipid molecule. The class II PITP, retinal degeneration type B (RdgB)α is a multi-domain protein and its PITP domain can bind and transfer PI and PA. In Drosophila photoreceptors, a well-defined G-protein-coupled phospholipase Cβ (PLCβ) signalling pathway, phototransduction defects resulting from loss of RdgBα can be rescued by expression of the PITP domain provided it is competent for both PI and PA transfer. We propose that RdgBα proteins maintain PI(4,5)P2 homoeostasis after PLC activation by facilitating the reciprocal transport of PA and PI at ER-PM membrane contact sites.

  17. Rab14 specifies the apical membrane through Arf6-mediated regulation of lipid domains and Cdc42

    PubMed Central

    Lu, Ruifeng; Wilson, Jean M.

    2016-01-01

    The generation of cell polarity is essential for the development of multi-cellular organisms as well as for the function of epithelial organs in the mature animal. Small GTPases regulate the establishment and maintenance of polarity through effects on cytoskeleton, membrane trafficking, and signaling. Using short-term 3-dimensional culture of MDCK cells, we find that the small GTPase Rab14 is required for apical membrane specification. Rab14 knockdown results in disruption of polarized lipid domains and failure of the Par/aPKC/Cdc42 polarity complex to localize to the apical membrane. These effects are mediated through tight control of lipid localization, as overexpression of the phosphatidylinositol 4-phosphate 5-kinase α [PtdIns(4)P5K] activator Arf6 or PtdIns(4)P5K alone, or treatment with the phosphatidylinositol 3-kinase (PtdInsI3K) inhibitor wortmannin, rescued the multiple-apical domain phenotype observed after Rab14 knockdown. Rab14 also co-immunoprecipitates and colocalizes with the small GTPase Cdc42, and Rab14 knockdown results in increased Cdc42 activity. Furthermore, Rab14 regulates trafficking of vesicles to the apical domain, mitotic spindle orientation, and midbody position, consistent with Rab14’s reported localization to the midbody as well as its effects upon Cdc42. These results position Rab14 at the top of a molecular cascade that regulates the establishment of cell polarity. PMID:27901125

  18. Specific interactions among transmembrane 4 superfamily (TM4SF) proteins and phosphoinositide 4-kinase.

    PubMed Central

    Yauch, R L; Hemler, M E

    2000-01-01

    In earlier work we established that phosphoinositide 4-kinase (PI 4-kinase) may associate with transmembrane 4 superfamily (TM4SF, tetraspanin) proteins, but critical specificity issues were not addressed. Here we demonstrate that at least five different TM4SF proteins (CD9, CD63, CD81, CD151 and A15/TALLA1) can associate with a similar or identical 55 kDa type II PI 4-kinase. These associations were specific, since we found no evidence for other phosphoinositide kinases (e.g. phosphoinositide 3-kinase and phosphoinositide-4-phosphate 5-kinase) associating with TM4SF proteins, and many other TM4SF proteins (including CD82 and CD53) did not associate with PI 4-kinase. CD63-PI 4-kinase complexes were almost entirely intracellular, and thus are distinct from other TM4SF-PI 4-kinase complexes (e.g. involving CD9), which are largely located in the plasma membrane. These results suggest that a specific subset of TM4SF proteins may recruit PI 4-kinase to specific membrane locations, and thereby influence phosphoinositide-dependent signalling. PMID:11042117

  19. Kinetics of PIP2 metabolism and KCNQ2/3 channel regulation studied with a voltage-sensitive phosphatase in living cells

    PubMed Central

    Falkenburger, Björn H.; Jensen, Jill B.

    2010-01-01

    The signaling phosphoinositide phosphatidylinositol 4,5-bisphosphate (PIP2) is synthesized in two steps from phosphatidylinositol by lipid kinases. It then interacts with KCNQ channels and with pleckstrin homology (PH) domains among many other physiological protein targets. We measured and developed a quantitative description of these metabolic and protein interaction steps by perturbing the PIP2 pool with a voltage-sensitive phosphatase (VSP). VSP can remove the 5-phosphate of PIP2 with a time constant of τ <300 ms and fully inhibits KCNQ currents in a similar time. PIP2 was then resynthesized from phosphatidylinositol 4-phosphate (PIP) quickly, τ = 11 s. In contrast, resynthesis of PIP2 after activation of phospholipase C by muscarinic receptors took ∼130 s. These kinetic experiments showed that (1) PIP2 activation of KCNQ channels obeys a cooperative square law, (2) the PIP2 residence time on channels is <10 ms and the exchange time on PH domains is similarly fast, and (3) the step synthesizing PIP2 by PIP 5-kinase is fast and limited primarily by a step(s) that replenishes the pool of plasma membrane PI(4)P. We extend the kinetic model for signaling from M1 muscarinic receptors, presented in our companion paper in this issue (Falkenburger et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.200910344), with this new information on PIP2 synthesis and KCNQ interaction. PMID:20100891

  20. Phosphorylation regulates the Star-PAP-PIPKIα interaction and directs specificity toward mRNA targets.

    PubMed

    Mohan, Nimmy; Sudheesh, A P; Francis, Nimmy; Anderson, Richard; Laishram, Rakesh S

    2015-08-18

    Star-PAP is a nuclear non-canonical poly(A) polymerase (PAP) that shows specificity toward mRNA targets. Star-PAP activity is stimulated by lipid messenger phosphatidyl inositol 4,5 bisphoshate (PI4,5P2) and is regulated by the associated Type I phosphatidylinositol-4-phosphate 5-kinase that synthesizes PI4,5P2 as well as protein kinases. These associated kinases act as coactivators of Star-PAP that regulates its activity and specificity toward mRNAs, yet the mechanism of control of these interactions are not defined. We identified a phosphorylated residue (serine 6, S6) on Star-PAP in the zinc finger region, the domain required for PIPKIα interaction. We show that S6 is phosphorylated by CKIα within the nucleus which is required for Star-PAP nuclear retention and interaction with PIPKIα. Unlike the CKIα mediated phosphorylation at the catalytic domain, Star-PAP S6 phosphorylation is insensitive to oxidative stress suggesting a signal mediated regulation of CKIα activity. S6 phosphorylation together with coactivator PIPKIα controlled select subset of Star-PAP target messages by regulating Star-PAP-mRNA association. Our results establish a novel role for phosphorylation in determining Star-PAP target mRNA specificity and regulation of 3'-end processing.

  1. The Saccharomyces cerevisiae flavodoxin-like proteins Ycp4 and Rfs1 play a role in stress response and in the regulation of genes related to metabolism.

    PubMed

    Cardona, Fernando; Orozco, Helena; Friant, Sylvie; Aranda, Agustín; del Olmo, Marcel lí

    2011-07-01

    SPI1 is a gene whose expression responds to many environmental stimuli, including entry into stationary phase. We have performed a screening to identify genes that activate SPI1 promoter when overexpressed. The phosphatidylinositol-4-phosphate 5-kinase gene MSS4 was identified as a positive activator of SPI1. Another SPI1 transcriptional regulator isolated was the flavodoxin-like gene YCP4. YCP4 and its homolog RFS1 regulate the expression of many genes during the late stages of growth. The double deletion mutant in YCP4 and its homolog RFS1 has an impact on gene expression related to metabolism by increasing the expression of genes involved in hexose transport and glycolysis, and decreasing expression of genes of amino acid metabolism pathways. Genes related to mating and response to pheromone show a decreased expression in the double mutant, while transcription of genes involved in translational elongation is increased. Deletion of these genes, together with the third member of the family, PST2, has a complex effect on the stress response. For instance, double mutant ycp4Δrfs1Δ has an increased response to oxidative stress, but a decreased tolerance to cell-damaging agent SDS. Additionally, this mutation affects chronological aging and slightly increases fermentative capacity.

  2. Phosphoinositide turnover in Toll-like receptor signaling and trafficking

    PubMed Central

    Tu Le, Oanh Thi; Ngoc Nguyen, Tu Thi; Lee, Sang Yoon

    2014-01-01

    Lipid components in biological membranes are essential for maintaining cellular function. Phosphoinositides, the phosphorylated derivatives of phosphatidylinositol (PI), regulate many critical cell processes involving membrane signaling, trafficking, and reorganization. Multiple metabolic pathways including phosphoinositide kinases and phosphatases and phospholipases tightly control spatio-temporal concentration of membrane phosphoinositides. Metabolizing enzymes responsible for PI 4,5-bisphosphate (PI(4,5)P2) production or degradation play a regulatory role in Toll-like receptor (TLR) signaling and trafficking. These enzymes include PI 4-phosphate 5-kinase, phosphatase and tensin homolog, PI 3-kinase, and phospholipase C. PI(4,5)P2 mediates the interaction with target cytosolic proteins to induce their membrane translocation, regulate vesicular trafficking, and serve as a precursor for other signaling lipids. TLR activation is important for the innate immune response and is implicated in diverse pathophysiological disorders. TLR signaling is controlled by specific interactions with distinct signaling and sorting adaptors. Importantly, TLR signaling machinery is differentially formed depending on a specific membrane compartment during signaling cascades. Although detailed mechanisms remain to be fully clarified, phosphoinositide metabolism is promising for a better understanding of such spatio-temporal regulation of TLR signaling and trafficking. [BMB Reports 2014; 47(7): 361-368] PMID:24856829

  3. Steering neuronal growth cones by shifting the imbalance between exocytosis and endocytosis.

    PubMed

    Tojima, Takuro; Itofusa, Rurika; Kamiguchi, Hiroyuki

    2014-05-21

    Extracellular molecular cues guide migrating growth cones along specific routes during development of axon tracts. Such processes rely on asymmetric elevation of cytosolic Ca(2+) concentrations across the growth cone that mediates its attractive or repulsive turning toward or away from the side with Ca(2+) elevation, respectively. Downstream of these Ca(2+) signals, localized activation of membrane trafficking steers the growth cone bidirectionally, with endocytosis driving repulsion and exocytosis causing attraction. However, it remains unclear how Ca(2+) can differentially regulate these opposite membrane-trafficking events. Here, we show that growth cone turning depends on localized imbalance between exocytosis and endocytosis and identify Ca(2+)-dependent signaling pathways mediating such imbalance. In embryonic chicken dorsal root ganglion neurons, repulsive Ca(2+) signals promote clathrin-mediated endocytosis through a 90 kDa splice variant of phosphatidylinositol-4-phosphate 5-kinase type-1γ (PIPKIγ90). In contrast, attractive Ca(2+) signals facilitate exocytosis but suppress endocytosis via Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and cyclin-dependent kinase 5 (Cdk5) that can inactivate PIPKIγ90. Blocking CaMKII or Cdk5 leads to balanced activation of both exocytosis and endocytosis that causes straight growth cone migration even in the presence of guidance signals, whereas experimentally perturbing the balance restores the growth cone's turning response. Remarkably, the direction of this resumed turning depends on relative activities of exocytosis and endocytosis, but not on the type of guidance signals. Our results suggest that navigating growth cones can be redirected by shifting the imbalance between exocytosis and endocytosis, highlighting the importance of membrane-trafficking imbalance for axon guidance and, possibly, for polarized cell migration in general.

  4. Involvement of PtdIns(4,5)P2 in the regulatory mechanism of small intestinal P-glycoprotein expression.

    PubMed

    Kobori, Takuro; Harada, Shinichi; Nakamoto, Kazuo; Tokuyama, Shogo

    2014-02-01

    Previously, we reported that repeated oral administration of etoposide (ETP) activates the ezrin/radixin/moesin (ERM) scaffold proteins for P-glycoprotein (P-gp) via Ras homolog gene family member A (RhoA)/Rho-associated coiled-coil containing protein kinase (ROCK) signaling, leading to increased ileal P-gp expression. Recent studies indicate that phosphatidyl inositol 4,5-bisphosphate [PtdIns(4,5)P2] regulates the plasma-membrane localization of certain proteins, and its synthase, the type I phosphatidyl inositol 4-phosphate 5-kinase (PI4P5K), is largely controlled by RhoA/ROCK. Here, we examined whether PtdIns(4,5)P2 and PI4P5K are involved in the increased expression of ileal P-gp following the ERM activation by ETP treatment. Male ddY mice (4-week-old) were treated with ETP (10 mg/kg/day, per os, p.o.) for 5 days. Protein-expression levels were measured by either western blot or dot blot analysis and molecular interactions were assessed using immunoprecipitation assays. ETP treatment significantly increased PI4P5K, ERM, and P-gp expression in the ileal membrane. This effect was suppressed following the coadministration of ETP with rosuvastatin (a RhoA inhibitor) or fasudil (a ROCK inhibitor). Notably, the PtdIns(4,5)P2 expression in the ileal membrane, as well as both P-gp and ERM levels coimmunoprecipitated with anti-PtdIns(4,5)P2 antibody, were increased by ETP treatment. PtdIns(4,5)P2 and PI4P5K may contribute to the increase in ileal P-gp expression observed following the ETP treatment, possibly through ERM activation via the RhoA/ROCK pathway.

  5. Phosphatidylinositol and phosphatidic acid transport between the ER and plasma membrane during PLC activation requires the Nir2 protein.

    PubMed

    Kim, Yeun Ju; Guzman-Hernandez, Maria Luisa; Wisniewski, Eva; Echeverria, Nicolas; Balla, Tamas

    2016-02-01

    Phospholipase C (PLC)-mediated hydrolysis of the limited pool of plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] requires replenishment from a larger pool of phosphatidylinositol (PtdIns) via sequential phosphorylation by PtdIns 4-kinases and phosphatidylinositol 4-phosphate (PtdIns4P) 5-kinases. Since PtdIns is synthesized in the endoplasmic reticulum (ER) and PtdIns(4,5)P2 is generated in the PM, it has been postulated that PtdIns transfer proteins (PITPs) provide the means for this lipid transfer function. Recent studies identified the large PITP protein, Nir2 as important for PtdIns transfer from the ER to the PM. It was also found that Nir2 was required for the transfer of phosphatidic acid (PtdOH) from the PM to the ER. In Nir2-depleted cells, activation of PLC leads to PtdOH accumulation in the PM and PtdIns synthesis becomes severely impaired. In quiescent cells, Nir2 is localized to the ER via interaction of its FFAT domain with ER-bound VAMP-associated proteins VAP-A and-B. After PLC activation, Nir2 also binds to the PM via interaction of its C-terminal domains with diacylglycerol (DAG) and PtdOH. Through these interactions, Nir2 functions in ER-PM contact zones. Mutations in VAP-B that have been identified in familial forms of amyotrophic lateral sclerosis (ALS or Lou-Gehrig's disease) cause aggregation of the VAP-B protein, which then impairs its binding to several proteins, including Nir2. These findings have shed new lights on the importance of non-vesicular lipid transfer of PtdIns and PtdOH in ER-PM contact zones with a possible link to a devastating human disease.

  6. Learning-related synaptic growth mediated by internalization of Aplysia cell adhesion molecule is controlled by membrane phosphatidylinositol 4,5-bisphosphate synthetic pathway.

    PubMed

    Lee, Seung-Hee; Shim, Jaehoon; Choi, Sun-Lim; Lee, Nuribalhae; Lee, Chang-Hoon; Bailey, Craig H; Kandel, Eric R; Jang, Deok-Jin; Kaang, Bong-Kiun

    2012-11-14

    Long-term facilitation in Aplysia is accompanied by the growth of new synaptic connections between the sensory and motor neurons of the gill-withdrawal reflex. One of the initial steps leading to the growth of these synapses is the internalization, induced by 5-HT, of the transmembrane isoform of Aplysia cell-adhesion molecule (TM-apCAM) from the plasma membrane of sensory neurons (Bailey et al., 1992). However, the mechanisms that govern the internalization of TM-apCAM and how this internalization is coupled to the molecular events that initiate the structural changes are not fully understood. Here, we report that the synthesis of membrane phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)], which is known to be mediated by a signaling cascade through Aplysia Sec7 protein (ApSec7) and phosphatidylinositol-4-phosphate 5-kinase type I α (PIP5KIα) is required for both the internalization of TM-apCAM and the initiation of synaptic growth during 5-HT-induced long-term facilitation. Pharmacological blockade of PI(4,5)P(2) synthesis by the application of the inhibitor phenylarsine oxide blocked the internalization of apCAM. Furthermore, perturbation of the endogenous activation of ApSec7 and its downstream target PIP5KIα also blocked 5-HT-mediated internalization of TM-apCAM and synaptic growth. Finally, long-term facilitation was specifically impaired by blocking the ApSec7 signaling pathway at sensory-to-motor neuron synapses. These data indicate that the ApSec7/PIP5KIα signaling pathway is actively recruited during learning-related 5-HT signaling and acts as a key regulator of apCAM internalization associated with the formation of new synaptic connections during long-term facilitation.

  7. Molecular basis for association of PIPKI gamma-p90 with clathrin adaptor AP-2.

    PubMed

    Kahlfeldt, Nina; Vahedi-Faridi, Ardeschir; Koo, Seong Joo; Schäfer, Johannes G; Krainer, Georg; Keller, Sandro; Saenger, Wolfram; Krauss, Michael; Haucke, Volker

    2010-01-22

    Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) is an essential determinant in clathrin-mediated endocytosis (CME). In mammals three type I phosphatidylinositol-4-phosphate 5-kinase (PIPK) enzymes are expressed, with the I gamma-p90 isoform being highly expressed in the brain where it regulates synaptic vesicle (SV) exo-/endocytosis at nerve terminals. How precisely PI(4,5)P(2) metabolism is controlled spatially and temporally is still uncertain, but recent data indicate that direct interactions between type I PIPK and components of the endocytic machinery, in particular the AP-2 adaptor complex, are involved. Here we demonstrated that PIPKI gamma-p90 associates with both the mu and beta2 subunits of AP-2 via multiple sites. Crystallographic data show that a peptide derived from the splice insert of the human PIPKI gamma-p90 tail binds to a cognate recognition site on the sandwich subdomain of the beta2 appendage. Partly overlapping aromatic and hydrophobic residues within the same peptide also can engage the C-terminal sorting signal binding domain of AP-2mu, thereby potentially competing with the sorting of conventional YXXØ motif-containing cargo. Biochemical and structure-based mutagenesis analysis revealed that association of the tail domain of PIPKI gamma-p90 with AP-2 involves both of these sites. Accordingly the ability of overexpressed PIPKI gamma tail to impair endocytosis of SVs in primary neurons largely depends on its association with AP-2 beta and AP-2mu. Our data also suggest that interactions between AP-2 and the tail domain of PIPKI gamma-p90 may serve to regulate complex formation and enzymatic activity. We postulate a model according to which multiple interactions between PIPKI gamma-p90 and AP-2 lead to spatiotemporally controlled PI(4,5)P(2) synthesis during clathrin-mediated SV endocytosis.

  8. Multiple New Loci Associated with Kidney Function and Chronic Kidney Disease: The CKDGen consortium

    PubMed Central

    Köttgen, Anna; Pattaro, Cristian; Böger, Carsten A.; Fuchsberger, Christian; Olden, Matthias; Glazer, Nicole L.; Parsa, Afshin; Gao, Xiaoyi; Yang, Qiong; Smith, Albert V.; O’Connell, Jeffrey R.; Li, Man; Schmidt, Helena; Tanaka, Toshiko; Isaacs, Aaron; Ketkar, Shamika; Hwang, Shih-Jen; Johnson, Andrew D.; Dehghan, Abbas; Teumer, Alexander; Paré, Guillaume; Atkinson, Elizabeth J.; Zeller, Tanja; Lohman, Kurt; Cornelis, Marilyn C.; Probst-Hensch, Nicole M.; Kronenberg, Florian; Tönjes, Anke; Hayward, Caroline; Aspelund, Thor; Eiriksdottir, Gudny; Launer, Lenore; Harris, Tamara B.; Rapmersaud, Evadnie; Mitchell, Braxton D.; Boerwinkle, Eric; Struchalin, Maksim; Cavalieri, Margherita; Singleton, Andrew; Giallauria, Francesco; Metter, Jeffery; de Boer, Ian; Haritunians, Talin; Lumley, Thomas; Siscovick, David; Psaty, Bruce M.; Zillikens, M. Carola; Oostra, Ben A.; Feitosa, Mary; Province, Michael; Levy, Daniel; de Andrade, Mariza; Turner, Stephen T.; Schillert, Arne; Ziegler, Andreas; Wild, Philipp S.; Schnabel, Renate B.; Wilde, Sandra; Muenzel, Thomas F.; Leak, Tennille S; Illig, Thomas; Klopp, Norman; Meisinger, Christa; Wichmann, H.-Erich; Koenig, Wolfgang; Zgaga, Lina; Zemunik, Tatijana; Kolcic, Ivana; Minelli, Cosetta; Hu, Frank B.; Johansson, Åsa; Igl, Wilmar; Zaboli, Ghazal; Wild, Sarah H; Wright, Alan F; Campbell, Harry; Ellinghaus, David; Schreiber, Stefan; Aulchenko, Yurii S; Rivadeneira, Fernando; Uitterlinden, Andre G; Hofman, Albert; Imboden, Medea; Nitsch, Dorothea; Brandstätter, Anita; Kollerits, Barbara; Kedenko, Lyudmyla; Mägi, Reedik; Stumvoll, Michael; Kovacs, Peter; Boban, Mladen; Campbell, Susan; Endlich, Karlhans; Völzke, Henry; Kroemer, Heyo K.; Nauck, Matthias; Völker, Uwe; Polasek, Ozren; Vitart, Veronique; Badola, Sunita; Parker, Alexander N.; Ridker, Paul M.; Kardia, Sharon L. R.; Blankenberg, Stefan; Liu, Yongmei; Curhan, Gary C.; Franke, Andre; Rochat, Thierry; Paulweber, Bernhard; Prokopenko, Inga; Wang, Wei; Gudnason, Vilmundur; Shuldiner, Alan R.; Coresh, Josef; Schmidt, Reinhold; Ferrucci, Luigi; Shlipak, Michael G.; van Duijn, Cornelia M.; Borecki, Ingrid; Krämer, Bernhard K.; Rudan, Igor; Gyllensten, Ulf; Wilson, James F.; Witteman, Jacqueline C.; Pramstaller, Peter P.; Rettig, Rainer; Hastie, Nick; Chasman, Daniel I.; Kao, W. H.; Heid, Iris M.; Fox, Caroline S.

    2010-01-01

    Chronic kidney disease (CKD) is a significant public health problem, and recent genetic studies have identified common CKD susceptibility variants. The CKDGen consortium performed a meta-analysis of genome-wide association data in 67,093 Caucasian individuals from 20 population-based studies to identify new susceptibility loci for reduced renal function, estimated by serum creatinine (eGFRcrea), cystatin C (eGFRcys), and CKD (eGFRcrea <60 ml/min/1.73m2; n = 5,807 CKD cases). Follow-up of the 23 genome-wide significant loci (p<5×10−8) in 22,982 replication samples identified 13 novel loci for renal function and CKD (in or near LASS2, GCKR, ALMS1, TFDP2, DAB2, SLC34A1, VEGFA, PRKAG2, PIP5K1B, ATXN2, DACH1, UBE2Q2, and SLC7A9) and 7 creatinine production and secretion loci (CPS1, SLC22A2, TMEM60, WDR37, SLC6A13, WDR72, BCAS3). These results further our understanding of biologic mechanisms of kidney function by identifying loci potentially influencing nephrogenesis, podocyte function, angiogenesis, solute transport, and metabolic functions of the kidney. PMID:20383146

  9. Plasticity-related Gene 5 (PRG5) Induces Filopodia and Neurite Growth and Impedes Lysophosphatidic Acid– and Nogo-A–mediated Axonal Retraction

    PubMed Central

    Broggini, Thomas; Nitsch, Robert

    2010-01-01

    Members of the plasticity-related gene (PRG1-4) family are brain-specific integral membrane proteins and implicated in neuronal plasticity, such as filopodia formation and axon growth after brain lesion. Here we report on the cloning of a novel member of the PRG family, PRG5, with high homologies to PRG3. PRG5 is regulated during brain and spinal cord development and is exclusively allocated within the nervous system. When introduced in neurons, PRG5 is distributed in the plasma membrane and induces filopodia as well as axon elongation and growth. Conversely, siRNA mediated knockdown of PRG5 impedes axon growth and disturbs filopodia formation. Here we show that PRG5 induces filopodia growth independently of Cdc42. Moreover, axon collapse and RhoA activation induced by LPA and myelin-associated neurite inhibitor Nogo-A is attenuated in the presence of PRG5, although direct activation of the RhoA-Rho-PIP5K kinase pathway abolishes PRG5 -formed neurites. Thus, we describe here the identification of a novel member of the PRG family that induces filopodia and axon elongation in a Cdc42-independent manner. In addition, PRG5 impedes brain injury-associated growth inhibitory signals upstream of the RhoA-Rho kinase pathway. PMID:20032306

  10. A Dibasic Amino Acid Pair Conserved in the Activation Loop Directs Plasma Membrane Localization and Is Necessary for Activity of Plant Type I/II Phosphatidylinositol Phosphate Kinase1[W

    PubMed Central

    Mikami, Koji; Saavedra, Laura; Hiwatashi, Yuji; Uji, Toshiki; Hasebe, Mitsuyasu; Sommarin, Marianne

    2010-01-01

    Phosphatidylinositol phosphate kinase (PIPK) is an enzyme involved in the regulation of cellular levels of phosphoinositides involved in various physiological processes, such as cytoskeletal organization, ion channel activation, and vesicle trafficking. In animals, research has focused on the modes of activation and function of PIPKs, providing an understanding of the importance of plasma membrane localization. However, it still remains unclear how this issue is regulated in plant PIPKs. Here, we demonstrate that the carboxyl-terminal catalytic domain, which contains the activation loop, is sufficient for plasma membrane localization of PpPIPK1, a type I/II B PIPK from the moss Physcomitrella patens. The importance of the carboxyl-terminal catalytic domain for plasma membrane localization was confirmed with Arabidopsis (Arabidopsis thaliana) AtPIP5K1. Our findings, in which substitution of a conserved dibasic amino acid pair in the activation loop of PpPIPK1 completely prevented plasma membrane targeting and abolished enzymatic activity, demonstrate its critical role in these processes. Placing our results in the context of studies of eukaryotic PIPKs led us to conclude that the function of the dibasic amino acid pair in the activation loop in type I/II PIPKs is plant specific. PMID:20427464

  11. Phosphoinositide 5- and 3-phosphatase activities of a voltage-sensing phosphatase in living cells show identical voltage dependence

    PubMed Central

    Keum, Dongil; Kim, Dong-Il; Suh, Byung-Chang

    2016-01-01

    Voltage-sensing phosphatases (VSPs) are homologs of phosphatase and tensin homolog (PTEN), a phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] 3-phosphatase. However, VSPs have a wider range of substrates, cleaving 3-phosphate from PI(3,4)P2 and probably PI(3,4,5)P3 as well as 5-phosphate from phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and PI(3,4,5)P3 in response to membrane depolarization. Recent proposals say these reactions have differing voltage dependence. Using Förster resonance energy transfer probes specific for different PIs in living cells with zebrafish VSP, we quantitate both voltage-dependent 5- and 3-phosphatase subreactions against endogenous substrates. These activities become apparent with different voltage thresholds, voltage sensitivities, and catalytic rates. As an analytical tool, we refine a kinetic model that includes the endogenous pools of phosphoinositides, endogenous phosphatase and kinase reactions connecting them, and four exogenous voltage-dependent 5- and 3-phosphatase subreactions of VSP. We show that apparent voltage threshold differences for seeing effects of the 5- and 3-phosphatase activities in cells are not due to different intrinsic voltage dependence of these reactions. Rather, the reactions have a common voltage dependence, and apparent differences arise only because each VSP subreaction has a different absolute catalytic rate that begins to surpass the respective endogenous enzyme activities at different voltages. For zebrafish VSP, our modeling revealed that 3-phosphatase activity against PI(3,4,5)P3 is 55-fold slower than 5-phosphatase activity against PI(4,5)P2; thus, PI(4,5)P2 generated more slowly from dephosphorylating PI(3,4,5)P3 might never accumulate. When 5-phosphatase activity was counteracted by coexpression of a phosphatidylinositol 4-phosphate 5-kinase, there was accumulation of PI(4,5)P2 in parallel to PI(3,4,5)P3 dephosphorylation

  12. Overexpression of the phosphatidylinositol synthase gene (ZmPIS) conferring drought stress tolerance by altering membrane lipid composition and increasing ABA synthesis in maize.

    PubMed

    Liu, Xiuxia; Zhai, Shumei; Zhao, Yajie; Sun, Baocheng; Liu, Cheng; Yang, Aifang; Zhang, Juren

    2013-05-01

    Phosphatidylinositol (PtdIns) synthase is a key enzyme in the phospholipid pathway and catalyses the formation of PtdIns. PtdIns is not only a structural component of cell membranes, but also the precursor of the phospholipid signal molecules that regulate plant response to environment stresses. Here, we obtained transgenic maize constitutively overexpressing or underexpressing PIS from maize (ZmPIS) under the control of a maize ubiquitin promoter. Transgenic plants were confirmed by PCR, Southern blotting analysis and real-time RT-PCR assay. The electrospray ionization tandem mass spectrometry (ESI-MS/MS)-based lipid profiling analysis showed that, under drought stress conditions, the overexpression of ZmPIS in maize resulted in significantly elevated levels of most phospholipids and galactolipids in leaves compared with those in wild type (WT). At the same time, the expression of some genes involved in the phospholipid metabolism pathway and the abscisic acid (ABA) biosynthesis pathway including ZmPLC, ZmPLD, ZmDGK1, ZmDGK3, ZmPIP5K9, ZmABA1, ZmNCED, ZmAAO1, ZmAAO2 and ZmSCA1 was markedly up-regulated in the overexpression lines after drought stress. Consistent with these results, the drought stress tolerance of the ZmPIS sense transgenic plants was enhanced significantly at the pre-flowering stages compared with WT maize plants. These results imply that ZmPIS regulates the plant response to drought stress through altering membrane lipid composition and increasing ABA synthesis in maize.

  13. Plasticity Related Gene 3 (PRG3) overcomes myelin-associated growth inhibition and promotes functional recovery after spinal cord injury

    PubMed Central

    Broggini, Thomas; Schnell, Lisa; Ghoochani, Ali; Mateos, José María; Buchfelder, Michael; Wiendieck, Kurt; Schäfer, Michael K.; Eyupoglu, Ilker Y.; Savaskan, Nicolai E.

    2016-01-01

    The Plasticity Related Gene family covers five, brain-specific, transmembrane proteins (PRG1-5, also termed LPPR1-5) that operate in neuronal plasticity during development, aging and brain trauma. Here we investigated the role of the PRG family on axonal and filopodia outgrowth. Comparative analysis revealed the strongest outgrowth induced by PRG3 (LPPR1). During development, PRG3 is ubiquitously located at the tip of neuronal processes and at the plasma membrane and declines with age. In utero electroporation of PRG3 induced dendritic protrusions and accelerated spine formations in cortical pyramidal neurons. The neurite growth promoting activity of PRG3 requires RasGRF1 (RasGEF1/Cdc25) mediated downstream signaling. Moreover, in axon collapse assays, PRG3-induced neurites resisted growth inhibitors such as myelin, Nogo-A (Reticulon/RTN-4), thrombin and LPA and impeded the RhoA-Rock-PIP5K induced neurite repulsion. Transgenic adult mice with constitutive PRG3 expression displayed strong axonal sprouting distal to a spinal cord lesion. Moreover, fostered PRG3 expression promoted complex motor-behavioral recovery compared to wild type controls as revealed in the Schnell swim test (SST). Thus, PRG3 emerges as a developmental RasGRF1-dependent conductor of filopodia formation and axonal growth enhancer. PRG3-induced neurites resist brain injury-associated outgrowth inhibitors and contribute to functional recovery after spinal cord lesions. Here, we provide evidence that PRG3 operates as an essential neuronal growth promoter in the nervous system. Maintaining PRG3 expression in aging brain may turn back the developmental clock for neuronal regeneration and plasticity. PMID:27744421

  14. Phylogenomics of phosphoinositide lipid kinases: perspectives on the evolution of second messenger signaling and drug discovery

    PubMed Central

    2011-01-01

    Background Phosphoinositide lipid kinases (PIKs) generate specific phosphorylated variants of phosatidylinositols (PtdIns) that are critical for second messenger signaling and cellular membrane remodeling. Mammals have 19 PIK isoforms spread across three major families: the PtIns 3-kinases (PI3Ks), PtdIns 4-kinases (PI4Ks), and PtdIns-P (PIP) kinases (PIPKs). Other eukaryotes have fewer yet varying PIK complements. PIKs are also an important, emerging class of drug targets for many therapeutic areas including cancer, inflammatory and metabolic diseases and host-pathogen interactions. Here, we report the genomic occurrences and evolutionary relationships or phylogenomics of all three PIK families across major eukaryotic groups and suggest potential ramifications for drug discovery. Results Our analyses reveal four core eukaryotic PIKs which are type III PIK4A and PIK4B, and at least one homolog each from PI3K (possibly PIK3C3 as the ancestor) and PIP5K families. We also applied evolutionary analyses to PIK disease ontology and drug discovery. Mutated PIK3CA are known to be oncogenic and several inhibitors are in anti-cancer clinical trials. We found conservation of activating mutations of PIK3CA in paralogous isoforms suggesting specific functional constraints on these residues. By mapping published compound inhibition data (IC50s) onto a phylogeny of PI3Ks, type II PI4Ks and distantly related, MTOR, ATM, ATR and PRKDC kinases, we also show that compound polypharmacology corresponds to kinase evolutionary relationships. Finally, we extended the rationale for drugs targeting PIKs of malarial Plasmodium falciparum, and the parasites, Leishmania sp. and Trypanosoma sp. by identifying those PIKs highly divergent from human homologs. Conclusion Our phylogenomic analysis of PIKs provides new insights into the evolution of second messenger signaling. We postulate two waves of PIK diversification, the first in metazoans with a subsequent expansion in cold

  15. Clinical genomics expands the morbid genome of intellectual disability and offers a high diagnostic yield.

    PubMed

    Anazi, S; Maddirevula, S; Faqeih, E; Alsedairy, H; Alzahrani, F; Shamseldin, H E; Patel, N; Hashem, M; Ibrahim, N; Abdulwahab, F; Ewida, N; Alsaif, H S; Al Sharif, H; Alamoudi, W; Kentab, A; Bashiri, F A; Alnaser, M; AlWadei, A H; Alfadhel, M; Eyaid, W; Hashem, A; Al Asmari, A; Saleh, M M; AlSaman, A; Alhasan, K A; Alsughayir, M; Al Shammari, M; Mahmoud, A; Al-Hassnan, Z N; Al-Husain, M; Osama Khalil, R; Abd El Meguid, N; Masri, A; Ali, R; Ben-Omran, T; El Fishway, P; Hashish, A; Ercan Sencicek, A; State, M; Alazami, A M; Salih, M A; Altassan, N; Arold, S T; Abouelhoda, M; Wakil, S M; Monies, D; Shaheen, R; Alkuraya, F S

    2017-04-01

    Intellectual disability (ID) is a measurable phenotypic consequence of genetic and environmental factors. In this study, we prospectively assessed the diagnostic yield of genomic tools (molecular karyotyping, multi-gene panel and exome sequencing) in a cohort of 337 ID subjects as a first-tier test and compared it with a standard clinical evaluation performed in parallel. Standard clinical evaluation suggested a diagnosis in 16% of cases (54/337) but only 70% of these (38/54) were subsequently confirmed. On the other hand, the genomic approach revealed a likely diagnosis in 58% (n=196). These included copy number variants in 14% (n=54, 15% are novel), and point mutations revealed by multi-gene panel and exome sequencing in the remaining 43% (1% were found to have Fragile-X). The identified point mutations were mostly recessive (n=117, 81%), consistent with the high consanguinity of the study cohort, but also X-linked (n=8, 6%) and de novo dominant (n=19, 13%). When applied directly on all cases with negative molecular karyotyping, the diagnostic yield of exome sequencing was 60% (77/129). Exome sequencing also identified likely pathogenic variants in three novel candidate genes (DENND5A, NEMF and DNHD1) each of which harbored independent homozygous mutations in patients with overlapping phenotypes. In addition, exome sequencing revealed de novo and recessive variants in 32 genes (MAMDC2, TUBAL3, CPNE6, KLHL24, USP2, PIP5K1A, UBE4A, TP53TG5, ATOH1, C16ORF90, SLC39A14, TRERF1, RGL1, CDH11, SYDE2, HIRA, FEZF2, PROCA1, PIANP, PLK2, QRFPR, AP3B2, NUDT2, UFC1, BTN3A2, TADA1, ARFGEF3, FAM160B1, ZMYM5, SLC45A1, ARHGAP33 and CAPS2), which we highlight as potential candidates on the basis of several lines of evidence, and one of these genes (SLC39A14) was biallelically inactivated in a potentially treatable form of hypermanganesemia and neurodegeneration. Finally, likely causal variants in previously published candidate genes were identified (ASTN1, HELZ, THOC6, WDR45B, ADRA

  16. Corneal dystrophies

    PubMed Central

    Klintworth, Gordon K

    2009-01-01

    The term corneal dystrophy embraces a heterogenous group of bilateral genetically determined non-inflammatory corneal diseases that are restricted to the cornea. The designation is imprecise but remains in vogue because of its clinical value. Clinically, the corneal dystrophies can be divided into three groups based on the sole or predominant anatomical location of the abnormalities. Some affect primarily the corneal epithelium and its basement membrane or Bowman layer and the superficial corneal stroma (anterior corneal dystrophies), the corneal stroma (stromal corneal dystrophies), or Descemet membrane and the corneal endothelium (posterior corneal dystrophies). Most corneal dystrophies have no systemic manifestations and present with variable shaped corneal opacities in a clear or cloudy cornea and they affect visual acuity to different degrees. Corneal dystrophies may have a simple autosomal dominant, autosomal recessive or X-linked recessive Mendelian mode of inheritance. Different corneal dystrophies are caused by mutations in the CHST6, KRT3, KRT12, PIP5K3, SLC4A11, TACSTD2, TGFBI, and UBIAD1 genes. Knowledge about the responsible genetic mutations responsible for these disorders has led to a better understanding of their basic defect and to molecular tests for their precise diagnosis. Genes for other corneal dystrophies have been mapped to specific chromosomal loci, but have not yet been identified. As clinical manifestations widely vary with the different entities, corneal dystrophies should be suspected when corneal transparency is lost or corneal opacities occur spontaneously, particularly in both corneas, and especially in the presence of a positive family history or in the offspring of consanguineous parents. Main differential diagnoses include various causes of monoclonal gammopathy, lecithin-cholesterol-acyltransferase deficiency, Fabry disease, cystinosis, tyrosine transaminase deficiency, systemic lysosomal storage diseases (mucopolysaccharidoses

  17. Synthesis of methylerythritol phosphate analogues and their evaluation as alternate substrates for IspDF and IspE from Agrobacterium tumefaciens.

    PubMed

    Krasutsky, Sergiy G; Urbansky, Marek; Davis, Chad E; Lherbet, Christian; Coates, Robert M; Poulter, C Dale

    2014-10-03

    The methylerythritol phosphate biosynthetic pathway, found in most Bacteria, some parasitic protists, and plant chloroplasts, converts D-glyceraldehyde phosphate and pyruvate to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), where it intersects with the mevalonate pathway found in some Bacteria, Archaea, and Eukarya, including the cytosol of plants. D-3-Methylerythritol-4-phosphate (MEP), the first pathway-specific intermediate in the pathway, is converted to IPP and DMAPP by the consecutive action of the IspD-H proteins. We synthesized five D-MEP analogues-D-erythritol-4-phosphate (EP), D-3-methylthrietol-4-phosphate (MTP), D-3-ethylerythritol-4-phosphate (EEP), D-1-amino-3-methylerythritol-4-phosphate (NMEP), and D-3-methylerythritol-4-thiolophosphate (MESP)-and studied their ability to function as alternative substrates for the reactions catalyzed by the IspDF fusion and IspE proteins from Agrobacterium tumefaciens, which covert MEP to the corresponding eight-membered cyclic diphosphate. All of the analogues, except MTP, and their products were substrates for the three consecutive enzymes.

  18. Synthesis of Methylerythritol Phosphate Analogues and Their Evaluation as Alternate Substrates for IspDF and IspE from Agrobacterium tumefaciens

    PubMed Central

    2015-01-01

    The methylerythritol phosphate biosynthetic pathway, found in most Bacteria, some parasitic protists, and plant chloroplasts, converts d-glyceraldehyde phosphate and pyruvate to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), where it intersects with the mevalonate pathway found in some Bacteria, Archaea, and Eukarya, including the cytosol of plants. d-3-Methylerythritol-4-phosphate (MEP), the first pathway-specific intermediate in the pathway, is converted to IPP and DMAPP by the consecutive action of the IspD-H proteins. We synthesized five d-MEP analogues—d-erythritol-4-phosphate (EP), d-3-methylthrietol-4-phosphate (MTP), d-3-ethylerythritol-4-phosphate (EEP), d-1-amino-3-methylerythritol-4-phosphate (NMEP), and d-3-methylerythritol-4-thiolophosphate (MESP)—and studied their ability to function as alternative substrates for the reactions catalyzed by the IspDF fusion and IspE proteins from Agrobacterium tumefaciens, which covert MEP to the corresponding eight-membered cyclic diphosphate. All of the analogues, except MTP, and their products were substrates for the three consecutive enzymes. PMID:25184438

  19. Identification of Candidate Zero Maintenance Paving Materials. Volume 1

    DTIC Science & Technology

    1977-05-01

    117 6.3.5 Sulfur Concrete ----------------------- 117 6.3.6 Sulfur-Foam----------------------------------------------- 118 6.4 Phosphate Cements...Sulfur-Admixture ----------------------------------- 120 6.5.4 Sulfur Concrete --------------------------------- i20 6.5.5 Sulfur-Foam...Sulfur Modified Asphalt Construction-- 112 6.1 Comparison of Mixture Proportions for Sulfur Concrete and Portland Cement Concrete (3000-psi at 24 hours

  20. Alterations in Hepatic and Aortic Phospholipase-C Coupled Receptors and Signal Transduction in Rat Intraperitoneal Sepsis

    DTIC Science & Technology

    1989-01-01

    phosphatidylinositol-4-phosphate, phosphatidyl- inositol or phosphatidic acid were noted (Figure 4). These results imply that not only is the receptor...PI hydrolysis. It is possible, however, that LPS in vivo is presented to the aortic smooth muscle cells in a way which is differenct from that in vitro...peritoneal macrophages. J Immun 134:526. Rosenbaum JS, Zera P, Umans VA, Ginsberg R, Hoffman BB (1986). Desensitization of aortic smooth muscle contraction in

  1. Hexameric assembly of the bifunctional methylerythritol 2,4-cyclodiphosphate synthase and protein-protein associations in the deoxy-xylulose-dependent pathway of isoprenoid precursor biosynthesis.

    PubMed

    Gabrielsen, Mads; Bond, Charles S; Hallyburton, Irene; Hecht, Stefan; Bacher, Adelbert; Eisenreich, Wolfgang; Rohdich, Felix; Hunter, William N

    2004-12-10

    The bifunctional methylerythritol 4-phosphate cytidylyltransferase methylerythritol 2,4-cyclodiphosphate synthase (IspDF) is unusual in that it catalyzes nonconsecutive reactions in the 1-deoxy-D-xylulose 5-phosphate (DOXP) pathway of isoprenoid precursor biosynthesis. The crystal structure of IspDF from the bacterial pathogen Campylobacter jejuni reveals an elongated hexamer with D3 symmetry compatible with the dimeric 2C-methyl-D-erythritol-4-phosphate cytidylyltransferase and trimeric 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase monofunctional enzymes. Complex formation of IspDF with 4-diphosphocytidyl-2C-methyl-D-erythritol kinase (IspE), the intervening enzyme activity in the pathway, has been observed in solution for the enzymes from C. jejuni and Agrobacterium tumefaciens. The monofunctional enzymes (2C-methyl-D-erythritol-4-phosphate cytidylyltransferase, IspE, and 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase) involved in the DOXP biosynthetic pathway of Escherichia coli also show physical associations. We propose that complex formation of the three enzymes at the core of the DOXP pathway can produce an assembly localizing 18 catalytic centers for the early stages of isoprenoid biosynthesis.

  2. Studies on the nonmevalonate pathway of terpene biosynthesis. The role of 2C-methyl-D-erythritol 2,4-cyclodiphosphate in plants.

    PubMed

    Fellermeier, M; Raschke, M; Sagner, S; Wungsintaweekul, J; Schuhr, C A; Hecht, S; Kis, K; Radykewicz, T; Adam, P; Rohdich, F; Eisenreich, W; Bacher, A; Arigoni, D; Zenk, M H

    2001-12-01

    2C-methyl-D-erythritol 2,4-cyclodiphosphate was recently shown to be formed from 2C-methyl-D-erythritol 4-phosphate by the consecutive action of IspD, IspE, and IspF proteins in the nonmevalonate pathway of terpenoid biosynthesis. To complement previous work with radiolabelled precursors, we have now demonstrated that [U-13C5]2C-methyl-D-erythritol 4-phosphate affords [U-13C5]2C-methyl-D-erythritol 2,4-cyclodiphosphate in isolated chromoplasts of Capsicum annuum and Narcissus pseudonarcissus. Moreover, chromoplasts are shown to efficiently convert 2C-methyl-D-erythritol 4-phosphate as well as 2C-methyl-D-erythritol 2,4-cyclodiphosphate into the carotene precursor phytoene. The bulk of the kinetic data collected in competition experiments with radiolabeled substrates is consistent with the notion that the cyclodiphosphate is an obligatory intermediate in the nonmevalonate pathway to terpenes. Studies with [2,2'-13C2]2C-methyl-D-erythritol 2,4-cyclodiphosphate afforded phytoene characterized by pairs of jointly transferred 13C atoms in the positions 17/1, 18/5, 19/9, and 20/13 and, at a lower abundance, in positions 16/1, 4/5, 8/9, and 12/13. A detailed scheme is presented for correlating the observed partial scrambling of label with the known lack of fidelity of the isopentenyl diphosphate/dimethylethyl diphosphate isomerase.

  3. Mice lacking p35, a neuronal specific activator of Cdk5, display cortical lamination defects, seizures, and adult lethality.

    PubMed

    Chae, T; Kwon, Y T; Bronson, R; Dikkes, P; Li, E; Tsai, L H

    1997-01-01

    The adult mammalian cortex is characterized by a distinct laminar structure generated through a well-defined pattern of neuronal migration. Successively generated neurons are layered in an "inside-out" manner to produce six cortical laminae. We demonstrate here that p35, the neuronal-specific activator of cyclin-dependent kinase 5, plays a key role in proper neuronal migration. Mice lacking p35, and thus p35/cdk5 kinase activity, display severe cortical lamination defects and suffer from sporadic adult lethality and seizures. Histological examination reveals that the mutant mice lack the characteristic laminated structure of the cortex. Neuronal birth-dating experiments indicate a reversed packing order of cortical neurons such that earlier born neurons reside in superficial layers and later generated neurons occupy deep layers. The phenotype of p35 mutant mice thus demonstrates that the formation of cortical laminar structure depends on the action of the p35/cdk5 kinase.

  4. High glucose increases Cdk5 activity in podocytes via transforming growth factor-β1 signaling pathway

    SciTech Connect

    Zhang, Yue; Li, Hongbo; Hao, Jun; Zhou, Yi; Liu, Wei

    2014-08-15

    Podocytes are highly specialized and terminally differentiated glomerular cells that play a vital role in the development and progression of diabetic nephropathy (DN). Cyclin-dependent kinase 5 (Cdk5), who is an atypical but essential member of the Cdk family of proline-directed serine/threonine kinases, has been shown as a key regulator of podocyte differentiation, proliferation and morphology. Our previous studies demonstrated that the expression of Cdk5 was significantly increased in podocytes of diabetic rats, and was closely related with podocyte injury of DN. However, the mechanisms of how expression and activity of Cdk5 are regulated under the high glucose environment have not yet been fully elucidated. In this study, we showed that high glucose up-regulated the expression of Cdk5 and its co-activator p35 with a concomitant increase in Cdk5 kinase activity in conditionally immortalized mouse podocytes in vitro. When exposed to 30 mM glucose, transforming growth factor-β1 (TGF-β1) was activated. Most importantly, we found that SB431542, the Tgfbr1 inhibitor, significantly decreased the expression of Cdk5 and p35 and Cdk5 kinase activity in high glucose-treated podocytes. Moreover, high glucose increased the expression of early growth response-1 (Egr-1) via TGF-β1-ERK1/2 pathway in podocytes and inhibition of Egr-1 by siRNA decreased p35 expression and Cdk5 kinase activity. Furthermore, inhibition of Cdk5 kinase activity effectively alleviated podocyte apoptosis induced by high glucose or TGF-β1. Thus, the TGF-β1-ERK1/2-Egr-1 signaling pathway may regulate the p35 expression and Cdk5 kinase activity in high glucose-treated podocytes, which contributes to podocyte injury of DN. - Highlights: • HG up-regulated the expression of Cdk5 and p35, and Cdk5 activity in podocytes. • HG activated TGF-β1 pathway and SB431542 inhibited Cdk5 expression and activity. • HG increased the expression of Egr-1 via TGF-β1-ERK1/2 pathway. • Inhibition of Egr-1

  5. Pathway and regulation of erythritol formation in Leuconostoc oenos.

    PubMed Central

    Veiga-da-Cunha, M; Santos, H; Van Schaftingen, E

    1993-01-01

    It was recently observed that Leuconostoc oenos GM, a wine lactic acid bacterium, produced erythritol anaerobically from glucose but not from fructose or ribose and that this production was almost absent in the presence of O2. In this study, the pathway of formation of erythritol from glucose in L. oenos was shown to involve the isomerization of glucose 6-phosphate to fructose 6-phosphate by a phosphoglucose isomerase, the cleavage of fructose 6-phosphate by a phosphoketolase, the reduction of erythrose 4-phosphate by an erythritol 4-phosphate dehydrogenase and, finally, the hydrolysis of erythritol 4-phosphate to erythritol by a phosphatase. Fructose 6-phosphate phosphoketolase was copurified with xylulose 5-phosphate phosphoketolase, and the activity of the latter was competitively inhibited by fructose 6-phosphate, with a Ki of 26 mM, corresponding to the Km of fructose 6-phosphate phosphoketolase (22 mM). These results suggest that the two phosphoketolase activities are borne by a single enzyme. Extracts of L. oenos were also found to contain NAD(P)H oxidase, which must be largely responsible for the reoxidation of NADPH and NADH in cells incubated in the presence of O2. In cells incubated with glucose, the concentrations of glucose 6-phosphate and of fructose 6-phosphate were higher in the absence of O2 than in its presence, explaining the stimulation by anaerobiosis of erythritol production. The increase in the hexose 6-phosphate concentration is presumably the result of a functional inhibition of glucose 6-phosphate dehydrogenase because of a reduction in the availability of NADP. PMID:8391532

  6. Effect of Enzyme Inhibitors on Terpene Trilactones Biosynthesis and Gene Expression Profiling in Ginkgo biloba Cultured Cells.

    PubMed

    Chen, Lijia; Tong, Hui; Wang, Mingxuan; Zhu, Jianhua; Zi, Jiachen; Song, Liyan; Yu, Rongmin

    2015-12-01

    The biosynthetic pathway of terpene trilactones of Ginkgo biloba is unclear. In this present study, suspension cultured cells of G. biloba were used to explore the regulation of the mevalonic acid (MVA) and methylerythritol 4-phosphate (MEP) pathways in response to specific enzyme inhibitors (lovastatin and clomazone). The results showed that the biosynthesis of bilobalide was more highly correlated with the MVA pathway, and the biosynthesis of ginkgolides was more highly correlated with the MEP pathway. Meanwhile, according to the results, it could be speculated that bilobalide might be a product of ginkgolide metabolism.

  7. Chemoenzymatic synthesis of 4-diphosphocytidyl-2-C-methyl-D-erythritol: A substrate for IspE.

    PubMed

    Narayanasamy, Prabagaran; Eoh, Hyungjin; Crick, Dean C

    2008-07-21

    Enantiomerically pure 2-C-methyl-D-erythritol 4-phosphate 1 (MEP) is synthesized from 1,2-O-isopropylidene-α-D-xylofuranose via facile benzylation in good yield. Subsequently, 1 is used for enzymatic synthesis of 4-diphosphocytidyl-2-C-methyl-D-erythritol 2 (CDP-ME) using 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (IspD). The chemoenzymatically synthesized 2 can be used as substrate for assay of IspE and for high throughput screening to identify IspE inhibitors.

  8. A spectrophotometric assay for the determination of 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase activity.

    PubMed

    Bernal, Cristobal; Mendez, Eva; Terencio, José; Boronat, Albert; Imperial, Santiago

    2005-05-15

    We report an assay for the determination of the activity of 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase, the enzyme which catalyzes the fourth reaction step of the 2-C-methyl-D-erythritol 4-phosphate pathway for the synthesis of isoprenoids, which is based on the spectrophotometrical determination of adenosine 5'-diphosphate using pyruvate kinase and L-lactate dehydrogenase as auxiliary enzymes. This method can be adapted to microtiter plates, can be automated, and because of its simplicity and speed can be useful for the functional characterization of the enzyme and for the screening of inhibitors with potential antibiotic or antimalarial action.

  9. A mutant pyruvate dehydrogenase E1 subunit allows survival of Escherichia coli strains defective in 1-deoxy-D-xylulose 5-phosphate synthase.

    PubMed

    Sauret-Güeto, Susanna; Urós, Eva María; Ibáñez, Ester; Boronat, Albert; Rodríguez-Concepción, Manuel

    2006-02-06

    The 2-C-methyl-D-erythritol 4-phosphate pathway has been proposed as a promising target to develop new antimicrobial agents. However, spontaneous mutations in Escherichia coli were observed to rescue the otherwise lethal loss of the first two enzymes of the pathway, 1-deoxy-D-xylulose 5-phosphate (DXP) synthase (DXS) and DXP reductoisomerase (DXR), with a relatively high frequency. A mutation in the gene encoding the E1 subunit of the pyruvate dehydrogenase complex was shown to be sufficient to rescue the lack of DXS but not DXR in vivo, suggesting that the mutant enzyme likely allows the synthesis of DXP or an alternative substrate for DXR.

  10. Molecular dynamics studies unravel role of conserved residues responsible for movement of ions into active site of DHBPS

    PubMed Central

    Shinde, Ranajit Nivrutti; Karthikeyan, Subramanian; Singh, Balvinder

    2017-01-01

    3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) catalyzes the conversion of D-ribulose 5-phosphate (Ru5P) to L-3,4-dihydroxy-2-butanone-4-phosphate in the presence of Mg2+. Although crystal structures of DHBPS in complex with Ru5P and non-catalytic metal ions have been reported, structure with Ru5P along with Mg2+ is still elusive. Therefore, mechanistic role played by Mg2+ in the structure of DHBPS is poorly understood. In this study, molecular dynamics simulations of DHBPS-Ru5P complex along with Mg2+ have shown entry of Mg2+ from bulk solvent into active site. Presence of Mg2+ in active site has constrained conformations of Ru5P and has reduced flexibility of loop-2. Formation of hydrogen bonds among Thr-108 and residues - Gly-109, Val-110, Ser-111, and Asp-114 are found to be critical for entry of Mg2+ into active site. Subsequent in silico mutations of residues, Thr-108 and Asp-114 have substantiated the importance of these interactions. Loop-4 of one monomer is being proposed to act as a “lid” covering the active site of other monomer. Further, the conserved nature of residues taking part in the transfer of Mg2+ suggests the same mechanism being present in DHBPS of other microorganisms. Thus, this study provides insights into the functioning of DHBPS that can be used for the designing of inhibitors. PMID:28079168

  11. Sac1--Vps74 structure reveals a mechanism to terminate phosphoinositide signaling in the Golgi apparatus

    SciTech Connect

    Cai, Yiying; Deng, Yongqiang; Horenkamp, Florian; Reinisch, Karin M.; Burd, Christopher G.

    2014-08-25

    Sac1 is a phosphoinositide phosphatase of the endoplasmic reticulum and Golgi apparatus that controls organelle membrane composition principally via regulation of phosphatidylinositol 4-phosphate signaling. We present a characterization of the structure of the N-terminal portion of yeast Sac1, containing the conserved Sac1 homology domain, in complex with Vps74, a phosphatidylinositol 4-kinase effector and the orthologue of human GOLPH3. The interface involves the N-terminal subdomain of the Sac1 homology domain, within which mutations in the related Sac3/Fig4 phosphatase have been linked to Charcot–Marie–Tooth disorder CMT4J and amyotrophic lateral sclerosis. Disruption of the Sac1–Vps74 interface results in a broader distribution of phosphatidylinositol 4-phosphate within the Golgi apparatus and failure to maintain residence of a medial Golgi mannosyltransferase. The analysis prompts a revision of the membrane-docking mechanism for GOLPH3 family proteins and reveals how an effector of phosphoinositide signaling serves a dual function in signal termination.

  12. Molecular dynamics studies unravel role of conserved residues responsible for movement of ions into active site of DHBPS

    NASA Astrophysics Data System (ADS)

    Shinde, Ranajit Nivrutti; Karthikeyan, Subramanian; Singh, Balvinder

    2017-01-01

    3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) catalyzes the conversion of D-ribulose 5-phosphate (Ru5P) to L-3,4-dihydroxy-2-butanone-4-phosphate in the presence of Mg2+. Although crystal structures of DHBPS in complex with Ru5P and non-catalytic metal ions have been reported, structure with Ru5P along with Mg2+ is still elusive. Therefore, mechanistic role played by Mg2+ in the structure of DHBPS is poorly understood. In this study, molecular dynamics simulations of DHBPS-Ru5P complex along with Mg2+ have shown entry of Mg2+ from bulk solvent into active site. Presence of Mg2+ in active site has constrained conformations of Ru5P and has reduced flexibility of loop-2. Formation of hydrogen bonds among Thr-108 and residues - Gly-109, Val-110, Ser-111, and Asp-114 are found to be critical for entry of Mg2+ into active site. Subsequent in silico mutations of residues, Thr-108 and Asp-114 have substantiated the importance of these interactions. Loop-4 of one monomer is being proposed to act as a “lid” covering the active site of other monomer. Further, the conserved nature of residues taking part in the transfer of Mg2+ suggests the same mechanism being present in DHBPS of other microorganisms. Thus, this study provides insights into the functioning of DHBPS that can be used for the designing of inhibitors.

  13. Sac1-Vps74 structure reveals a mechanism to terminate phosphoinositide signaling in the Golgi apparatus.

    PubMed

    Cai, Yiying; Deng, Yongqiang; Horenkamp, Florian; Reinisch, Karin M; Burd, Christopher G

    2014-08-18

    Sac1 is a phosphoinositide phosphatase of the endoplasmic reticulum and Golgi apparatus that controls organelle membrane composition principally via regulation of phosphatidylinositol 4-phosphate signaling. We present a characterization of the structure of the N-terminal portion of yeast Sac1, containing the conserved Sac1 homology domain, in complex with Vps74, a phosphatidylinositol 4-kinase effector and the orthologue of human GOLPH3. The interface involves the N-terminal subdomain of the Sac1 homology domain, within which mutations in the related Sac3/Fig4 phosphatase have been linked to Charcot-Marie-Tooth disorder CMT4J and amyotrophic lateral sclerosis. Disruption of the Sac1-Vps74 interface results in a broader distribution of phosphatidylinositol 4-phosphate within the Golgi apparatus and failure to maintain residence of a medial Golgi mannosyltransferase. The analysis prompts a revision of the membrane-docking mechanism for GOLPH3 family proteins and reveals how an effector of phosphoinositide signaling serves a dual function in signal termination.

  14. Metabolic and structural evidence for the existence of a third species of polyphosphoinositide in cells: D-phosphatidyl-myo-inositol 3-phosphate.

    PubMed Central

    Stephens, L; Hawkins, P T; Downes, C P

    1989-01-01

    When human 1321 N1 astrocytoma cells were labelled to steady state with [3H]inositol and briefly with [32P]orthophosphate, a compound which contained both radiotracers and which co-migrated with phosphatidylinositol-myo-inositol 4-phosphate during t.l.c. could be extracted in acidic chloroform/methanol. Treatment with methylamine under conditions which lead to deacylation of conventional glycerophospholipids yielded a water-soluble moiety which was labelled with both radioisotopes and was eluted from an anion-exchange h.p.l.c. column with a retention time similar to, but distinct from, that of glycerophosphoinositol 4-phosphate. Experiments using sodium periodate and selective phosphatase enzymes to degrade this compound systematically generated a series of products which suggested the structure of the parent phospholipid was phosphatidyl-myo-inositol 3-phosphate (PtdIns3P). PtdIns3P is metabolically closely related to the pool(s) of inositol phospholipid(s) that serves as substrate(s) for an agonist-sensitive phosphoinositidase C, as the levels of PtdIns3P fell significantly when 1321 N1 cells were stimulated with carbachol. The relative rate of turnover of the inositol moiety of PtdIns3P is similar to that of both of the major polyphosphoinositides and significantly higher than that of total cellular phosphatidyl-myo-inositol. This suggests that all three polyphosphoinositides are synthesized from a common, rapidly metabolized, pool of phosphatidyl-myo-inositol. PMID:2541684

  15. The large GTPase dynamin regulates actin comet formation and movement in living cells

    PubMed Central

    Orth, James D.; Krueger, E. W.; Cao, H.; McNiven, Mark A.

    2002-01-01

    The large GTPase dynamin (Dyn2) has been demonstrated by us and others to interact with several different actin-binding proteins. To define how Dyn2 might participate in actin dynamics in livings cells we have expressed green fluorescent protein (GFP)-tagged Dyn2 in cultured cells and observed labeling of comet-like vesicles and macropinosomes. The comet structures progressed with a constant velocity and were reminiscent of actin comets associated with motile vesicles in cells expressing type I phosphatidylinositol phosphate 5-kinases. Based on these observations we sought to determine whether Dyn2 is an integral component of actin comets. Cells expressing type I phosphatidylinositol phosphate 5-kinase and Dyn2-GFP revealed a prominent colocalization of Dyn2 and actin in comet structures. Interestingly, comet formation and motility were normal in cells expressing wild-type Dyn2-GFP but altered markedly in Dyn2 mutant-expressing cells. Dyn2K44A-GFP mutant cells displayed a significant reduction in comet number, length, velocity, and efficiency of movement. In contrast, comets in cells expressing Dyn2ΔPRD-GFP appeared dark and did not incorporate the mutant Dyn2 protein, indicating that the proline-rich domain (PRD) is required for Dyn2 recruitment. Further, these comets were significantly longer and slower than those in control cells. These findings demonstrate a role for Dyn2 in actin-based vesicle motility. PMID:11782546

  16. Loss-of-function and gain-of-function mutations in FAB1A/B impair endomembrane homeostasis, conferring pleiotropic developmental abnormalities in Arabidopsis.

    PubMed

    Hirano, Tomoko; Matsuzawa, Tomohiko; Takegawa, Kaoru; Sato, Masa H

    2011-02-01

    In eukaryotic cells, PtdIns 3,5-kinase, Fab1/PIKfyve produces PtdIns (3,5) P(2) from PtdIns 3-P, and functions in vacuole/lysosome homeostasis. Herein, we show that expression of Arabidopsis (Arabidopsis thaliana) FAB1A/B in fission yeast (Schizosaccharomyces pombe) fab1 knockout cells fully complements the vacuole morphology phenotype. Subcellular localizations of FAB1A and FAB1B fused with green fluorescent protein revealed that FAB1A/B-green fluorescent proteins localize to the endosomes in root epidermal cells of Arabidopsis. Furthermore, reduction in the expression levels of FAB1A/B by RNA interference impairs vacuolar acidification and endocytosis. These results indicate that Arabidopsis FAB1A/B functions as PtdIns 3,5-kinase in plants and in fission yeast. Conditional knockdown mutant shows various phenotypes including root growth inhibition, hyposensitivity to exogenous auxin, and disturbance of root gravitropism. These phenotypes are observed also in the overproducing mutants of FAB1A and FAB1B. The overproducing mutants reveal additional morphological phenotypes including dwarfism, male-gametophyte sterility, and abnormal floral organs. Taken together, this evidence indicates that imbalanced expression of FAB1A/B impairs endomembrane homeostasis including endocytosis, vacuole formation, and vacuolar acidification, which causes pleiotropic developmental phenotypes mostly related to the auxin signaling in Arabidopsis.

  17. Discovery of a novel allosteric inhibitor-binding site in ERK5: comparison with the canonical kinase hinge ATP-binding site

    PubMed Central

    Chen, Hongming; Tucker, Julie; Wang, Xiaotao; Gavine, Paul R.; Phillips, Chris; Augustin, Martin A.; Schreiner, Patrick; Steinbacher, Stefan; Preston, Marian; Ogg, Derek

    2016-01-01

    MAP kinases act as an integration point for multiple biochemical signals and are involved in a wide variety of cellular processes such as proliferation, differentiation, regulation of transcription and development. As a member of the MAP kinase family, ERK5 (MAPK7) is involved in the downstream signalling pathways of various cell-surface receptors, including receptor tyrosine kinases and G protein-coupled receptors. In the current study, five structures of the ERK5 kinase domain co-crystallized with ERK5 inhibitors are reported. Interestingly, three of the compounds bind at a novel allosteric binding site in ERK5, while the other two bind at the typical ATP-binding site. Binding of inhibitors at the allosteric site is accompanied by displacement of the P-loop into the ATP-binding site and is shown to be ATP-competitive in an enzymatic assay of ERK5 kinase activity. Kinase selectivity data show that the most potent allosteric inhibitor exhibits superior kinase selectivity compared with the two inhibitors that bind at the canonical ATP-binding site. An analysis of these structures and comparison with both a previously published ERK5–inhibitor complex structure (PDB entry 4b99) and the structures of three other kinases (CDK2, ITK and MEK) in complex with allosteric inhibitors are presented. PMID:27139631

  18. Carbohydrate-binding domain of the POMGnT1 stem region modulates O-mannosylation sites of α-dystroglycan

    PubMed Central

    Kuwabara, Naoyuki; Manya, Hiroshi; Yamada, Takeyuki; Tateno, Hiroaki; Kanagawa, Motoi; Kobayashi, Kazuhiro; Akasaka-Manya, Keiko; Hirose, Yuriko; Mizuno, Mamoru; Ikeguchi, Mitsunori; Toda, Tatsushi; Hirabayashi, Jun; Senda, Toshiya; Endo, Tamao; Kato, Ryuichi

    2016-01-01

    The dystrophin glycoprotein complex, which connects the cell membrane to the basement membrane, is essential for a variety of biological events, including maintenance of muscle integrity. An O-mannose–type GalNAc-β1,3-GlcNAc-β1,4-(phosphate-6)-Man structure of α-dystroglycan (α-DG), a subunit of the complex that is anchored to the cell membrane, interacts directly with laminin in the basement membrane. Reduced glycosylation of α-DG is linked to some types of inherited muscular dystrophy; consistent with this relationship, many disease-related mutations have been detected in genes involved in O-mannosyl glycan synthesis. Defects in protein O-linked mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGnT1), a glycosyltransferase that participates in the formation of GlcNAc-β1,2-Man glycan, are causally related to muscle-eye-brain disease (MEB), a congenital muscular dystrophy, although the role of POMGnT1 in postphosphoryl modification of GalNAc-β1,3-GlcNAc-β1,4-(phosphate-6)-Man glycan remains elusive. Our crystal structures of POMGnT1 agreed with our previous results showing that the catalytic domain recognizes substrate O-mannosylated proteins via hydrophobic interactions with little sequence specificity. Unexpectedly, we found that the stem domain recognizes the β-linked GlcNAc of O-mannosyl glycan, an enzymatic product of POMGnT1. This interaction may recruit POMGnT1 to a specific site of α-DG to promote GlcNAc-β1,2-Man clustering and also may recruit other enzymes that interact with POMGnT1, e.g., fukutin, which is required for further modification of the GalNAc-β1,3-GlcNAc-β1,4-(phosphate-6)-Man glycan. On the basis of our findings, we propose a mechanism for the deficiency in postphosphoryl modification of the glycan observed in POMGnT1-KO mice and MEB patients. PMID:27493216

  19. Phosphatidylinositol 4-kinases: Function, structure, and inhibition

    SciTech Connect

    Boura, Evzen Nencka, Radim

    2015-10-01

    The phosphatidylinositol 4-kinases (PI4Ks) synthesize phosphatidylinositol 4-phosphate (PI4P), a key member of the phosphoinositide family. PI4P defines the membranes of Golgi and trans-Golgi network (TGN) and regulates trafficking to and from the Golgi. Humans have two type II PI4Ks (α and β) and two type III enzymes (α and β). Recently, the crystal structures were solved for both type II and type III kinase revealing atomic details of their function. Importantly, the type III PI4Ks are hijacked by +RNA viruses to create so-called membranous web, an extensively phosphorylated and modified membrane system dedicated to their replication. Therefore, selective and potent inhibitors of PI4Ks have been developed as potential antiviral agents. Here we focus on the structure and function of PI4Ks and their potential in human medicine.

  20. Studies on RBC lipid and protein phosphorylation during blood bank storage

    SciTech Connect

    Dumaswala, U.J.; Bryan, D.J.; Greenwalt, T.J.

    1986-05-01

    Recent evidence has suggested that phosphoinositides play a significant role in maintaining membrane structure and function. Their importance during blood bank storage is not understood. They have performed preliminary studies of the phosphoinositide synthetic pathway enzymes of RBC during blood bank storage. At 0 and 35 days of storage leaky ghosts were prepared and incubated with (..gamma..-/sup 32/P)ATP for 5 minutes at 30 C. One aliquot was subjected to acidified solvent extraction and thin layer chromatography. The labeled phosphoinositide -4,5 biphosphate (PIP/sub 2/), phosphoinositide-4 phosphate (PIP) and phosphatidic acid (PA) spots were scraped and counted by liquid scintillation spectrometry. Another aliquot was used for SDS-PAGE and the radioactivity associated with the ..beta..-spectrin was measured. These experiments suggest a decrease in RBC phosphoinositol and PIP-Kinases and ..beta..-spectrin kinase activities during blood bank storage. Further studies are being done to evaluate significance of these observations.

  1. Wear and friction of oxidation-resistant mechanical carbon graphites at 650 C in air

    NASA Technical Reports Server (NTRS)

    Allen, G. P.; Wisnader, D. W.

    1975-01-01

    Studies were conducted to determine the friction and wear properties of experimental carbon-graphites. Hemispherically tipped carbon-graphite rider specimens were tested in sliding contact with rotating Inconel X-750 disks in air. A surface speed of 1.33 m/sec, a load of 500 g, and a specimen temperature of 650 C were used. Results indicate: (1) hardness is not a major factor in determining friction and wear under the conditions of these studies. (2) Friction and wear as low as or lower than those observed for a good commercial seal material were attained with some of the experimental materials studied. (3) The inclusion of boron carbide (as an oxidation inhibitor) has a strong influence on wear rate. (4) Phosphate treatment reduces the friction coefficient when boron carbide is not present in the base material.

  2. Babesia gibsoni: detection in blood smears and formalin-fixed, paraffin-embedded tissues using deoxyribonucleic acid in situ hybridization analysis.

    PubMed

    Yamasaki, Masahiro; Kobayashi, Yusuke; Nakamura, Kensuke; Sasaki, Noboru; Murakami, Masahiro; Rajapakshage, Bandula Kumara Wickramasekara; Ohta, Hiroshi; Yamato, Osamu; Maede, Yoshimitsu; Takiguchi, Mitsuyoshi

    2011-01-01

    In this study, we attempted to detect Babesia gibsoni in blood smears and formalin-fixed, paraffin-embedded tissues obtained from B. gibsoni-infected dogs using in situ hybridization. Using a digoxigenin-conjugated deoxyribonucleic acid (DNA) probe, both intraerythrocytic and exoerythrocytic parasites in the culture could be specifically stained in blood smears fixed with 4% phosphate-buffered paraformaldehyde. This indicated that genomic DNA extracted from the parasites could be detected using in situ hybridization. Moreover, the parasite could be specifically stained in paraffin-embedded spleen, lymph node, and kidney sections using in situ hybridization. Infected erythrocytes in blood vessels in the spleen and kidney, hemosiderin-laden macrophages in the spleen, and phagocytized erythrocytes, which seemed to be infected with the parasites, in lymph nodes were also specifically stained. This suggests that in situ hybridization can be utilized to investigate both the life cycle of B. gibsoni and the pathological condition of canine babesiosis.

  3. Isoprenoid biosynthesis in higher plants and in Escherichia coli: on the branching in the methylerythritol phosphate pathway and the independent biosynthesis of isopentenyl diphosphate and dimethylallyl diphosphate.

    PubMed Central

    Hoeffler, Jean-François; Hemmerlin, Andréa; Grosdemange-Billiard, Catherine; Bach, Thomas J; Rohmer, Michel

    2002-01-01

    In the bacterium Escherichia coli, the mevalonic-acid (MVA)-independent 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway is characterized by two branches leading separately to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). The signature of this branching is the retention of deuterium in DMAPP and the deuterium loss in IPP after incorporation of 1-[4-(2)H]deoxy-d-xylulose ([4-(2)H]DX). Feeding tobacco BY-2 cell-suspension cultures with [4-(2)H]DX resulted in deuterium retention in the isoprene units derived from DMAPP, as well as from IPP in the plastidial isoprenoids, phytoene and plastoquinone, synthesized via the MEP pathway. This labelling pattern represents direct evidence for the presence of the DMAPP branch of the MEP pathway in a higher plant, and shows that IPP can be synthesized from DMAPP in plant plastids, most probably via a plastidial IPP isomerase. PMID:12010124

  4. Specificity determinants in phosphoinositide dephosphorylation: crystal structure of an archetypal inositol polyphosphate 5-phosphatase.

    PubMed

    Tsujishita, Y; Guo, S; Stolz, L E; York, J D; Hurley, J H

    2001-05-04

    Inositol polyphosphate 5-phosphatases are central to intracellular processes ranging from membrane trafficking to Ca(2+) signaling, and defects in this activity result in the human disease Lowe syndrome. The 1.8 resolution structure of the inositol polyphosphate 5-phosphatase domain of SPsynaptojanin bound to Ca(2+) and inositol (1,4)-bisphosphate reveals a fold and an active site His and Asp pair resembling those of several Mg(2+)-dependent nucleases. Additional loops mediate specific inositol polyphosphate contacts. The 4-phosphate of inositol (1,4)-bisphosphate is misoriented by 4.6 compared to the reactive geometry observed in the apurinic/apyrimidinic endonuclease 1, explaining the dephosphorylation site selectivity of the 5-phosphatases. Based on the structure, a series of mutants are described that exhibit altered substrate specificity providing general determinants for substrate recognition.

  5. Transcript Quantification of Genes Involved in Steviol Glycoside Biosynthesis in Stevia rebaudiana Bertoni by Real-Time Polymerase Chain Reaction (RT-PCR).

    PubMed

    Modi, Arpan; Kumar, Nitish; Narayanan, Subhash

    2016-01-01

    Stevia (Stevia rebaudiana Bertoni) is a medicinal plant having sweet, diterpenoid glycosides known as steviol glycosides which are 200-300 times sweeter than sucrose (0.4 % solution). They are synthesized mainly in the leaves via plastid localized 2-C-methyl-D-erythrose-4-phosphate pathway (MEP pathway). Fifteen genes are involved in the formation of these glycosides. In the present protocol, a method for the quantification of transcripts of these genes is shown. The work involves RNA extraction and cDNA preparation, and therefore, procedures for the confirmation of DNA-free cDNA preparation have also been illustrated. Moreover, details of plant treatments are not mentioned as this protocol may apply to relative gene expression profile in any medicinal plant with any treatment. The treatments are numbered as T0 (Control), T1, T2, T3, and T4.

  6. Structural basis for phosphoinositide substrate recognition, catalysis, and membrane interactions in human inositol polyphosphate 5-phosphatases.

    PubMed

    Trésaugues, Lionel; Silvander, Camilla; Flodin, Susanne; Welin, Martin; Nyman, Tomas; Gräslund, Susanne; Hammarström, Martin; Berglund, Helena; Nordlund, Pär

    2014-05-06

    SHIP2, OCRL, and INPP5B belong to inositol polyphosphate 5-phophatase subfamilies involved in insulin regulation and Lowes syndrome. The structural basis for membrane recognition, substrate specificity, and regulation of inositol polyphosphate 5-phophatases is still poorly understood. We determined the crystal structures of human SHIP2, OCRL, and INPP5B, the latter in complex with phosphoinositide substrate analogs, which revealed a membrane interaction patch likely to assist in sequestering substrates from the lipid bilayer. Residues recognizing the 1-phosphate of the substrates are highly conserved among human family members, suggesting similar substrate binding modes. However, 3- and 4-phosphate recognition varies and determines individual substrate specificity profiles. The high conservation of the environment of the scissile 5-phosphate suggests a common reaction geometry for all members of the human 5-phosphatase family.

  7. New potential solutions for the chemolysis of urinary phosphate calculi determined by an in vitro study.

    PubMed

    Zhang, Jinqing; Wang, Shuo; Hong, Jingfan; Liu, Chunxiao; Jiang, Yanbin

    2015-04-01

    To find a more efficient solution for chemolysis of urinary calculi, several organic acids were chosen to form solutions by consulting the composition of a classic solution, Suby G. The solutions together with Renacidin, another classic solution, were designed to react with the 4 phosphate components of urinary stone. The processes were real-time measured and analysed by a focused beam reflectance measurement, and the efficiency factors were investigated and discussed in detail. The results show that several organic acids, e.g. hydroxyacetic acid, lactic acid and α-ketoglutaric acid, are more efficient than citric acid in dissolving urinary phosphate calculus. The new solutions containing the organic acids are promising for improving chemolysis treatment.

  8. Differential incorporation of 1-deoxy-D-xylulose into (3S)-linalool and geraniol in grape berry exocarp and mesocarp.

    PubMed

    Luan, Fang; Wüst, Matthias

    2002-07-01

    In vivo feeding experiments with [5,5-(2)H(2)]mevalonic acid lactone (MVL) and [5,5-(2)H(2)]-1-deoxy-D-xylulose (DOX) indicate that the novel mevalonate-independent 1-deoxy- D-xylulose 5-phosphate/2C-methyl- D-erythritol 4-phosphate (DOXP/MEP) pathway is the dominant metabolic route for monoterpene biosynthesis in grape berry exocarp and mesocarp and in grape leaves. The highly uneven distribution of the monoterpene alcohols (3S)-linalool and geraniol between leaves, berry exocarp and berry mesocarp can be attributed to a compartmentation of monoterpene metabolism. In grape berries incorporation of [5,5-(2)H(2)]-DOX into geraniol is mainly restricted to the exocarp, whereas (3S)-linalool biosynthesis can be detected in exocarp as well as in mesocarp tissue. The results demonstrate that grape berries exhibit an autonomic monoterpene biosynthesis via the novel DOXP/MEP route throughout the ripening process.

  9. Validation of a homology model of Mycobacterium tuberculosis DXS: rationalization of observed activities of thiamine derivatives as potent inhibitors of two orthologues of DXS.

    PubMed

    Masini, T; Lacy, B; Monjas, L; Hawksley, D; de Voogd, A R; Illarionov, B; Iqbal, A; Leeper, F J; Fischer, M; Kontoyianni, M; Hirsch, A K H

    2015-12-14

    The enzyme DXS catalyzes the first, rate-limiting step of the 2-C-methyl-d-erythritol-4-phosphate (MEP, 1) pathway using thiamine diphosphate (ThDP) as cofactor; the DXS-catalyzed reaction constitutes also the first step in vitamin B1 and B6 metabolism in bacteria. DXS is the least studied among the enzymes of this pathway in terms of crystallographic information, with only one complete crystal structure deposited in the Protein Data Bank (Deinococcus radiodurans DXS, PDB: ). We synthesized a series of thiamine and ThDP derivatives and tested them for their biochemical activity against two DXS orthologues, namely D. radiodurans DXS and Mycobacterium tuberculosis DXS. These experimental results, combined with advanced docking studies, led to the development and validation of a homology model of M. tuberculosis DXS, which, in turn, will guide medicinal chemists in rationally designing potential inhibitors for M. tuberculosis DXS.

  10. Purification and Characterization of Pea Chloroplastic Phosphoriboisomerase 12

    PubMed Central

    Skrukrud, Cynthia L.; Gordon, Ilana M.; Dorwin, Sally; Yuan, Xiao-Hua; Johansson, Göte; Anderson, Louise E.

    1991-01-01

    Pea (Pisum sativum L.) chloroplastic phosphoriboisomerase (EC 5.3.1.6) can be purified to apparent homogeneity in less than 2 days time with a 53% yield. Important steps in the purification include heat treatment and pseudoaffinity chromatography on Red H-3BN Sepharose. The purified isomerase has a subunit molecular mass of 26.4 kD. The N-terminal sequence has been determined through 34 residues. pH optima are 7.8 (ribose-5-phosphate) and 7.7 (ribulose-5-phosphate); Km values are 0.9 millimolar (ribose-5-phosphate) and 0.6 millimolar (ribulose-5-phosphate). The enzyme is inhibited by erythrose-4-phosphate, sedoheptulosebisphosphate, glyceraldehyde-3-phosphate, and 3-phosphoglycerate at concentrations close to those found in photosynthesizing chloroplasts. Countercurrent phase partitioning experiments indicate that the pea chloroplastic phosphoriboisomerase interacts physically with phosphoribulokinase. ImagesFigure 1 PMID:16668459

  11. Light-stimulated inositolphospholipid turnover in Samanea saman leaf pulvini

    SciTech Connect

    Morse, M.J.; Crain, R.C.; Satter, R.L.

    1987-10-01

    Leaflets of Samanea saman open and close rhythmically, driven by an endogenous circadian clock. Light has a rapid, direct effect on the movements and also rephases the rhythm. The authors investigated whether light signals might be mediated by increased inositolphospholipid turnover, a mechanism for signal transduction that is widely utilized in animal systems. Samanea motor organs (pulvini) labeled with (/sup 3/H)inositol were irradiated briefly (5-30 sec) with white light, and membrane-localized phosphatidylinositol phosphates and their aqueous breakdown products, the inositol phosphates, were examined. After a 15-sec or longer light pulse, labeled phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate decreased and their labeled metabolic products inositol 1,4-biphosphate and inositol 1,4,5-trisphosphate increased changes characteristic of inositolphospholipid turnover. The authors conclude that inositolphospholipid turnover may act as a phototransduction mechanism in Samanea pulvini in a manner that is similar to that reported in animal systems.

  12. The inositol phosphate/diacylglycerol signalling pathway in Trypanosoma cruzi.

    PubMed Central

    Docampo, R; Pignataro, O P

    1991-01-01

    Using [32P]Pi and [3H]inositol as precursors, we have detected the presence of phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, and their derivatives inositol phosphate, inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate respectively, in Trypanosoma cruzi epimastigotes. Using digitonin-permeabilized cells it was possible to detect a stimulation in the formation of inositol 1,4,5-trisphosphate and inositol 1,4-bisphosphate as well as an increased generation of diacylglycerol in the presence of 1 mM-CaCl2. These results are consistent with the operation of a functional inositol phosphate/diacylglycerol pathway in T. cruzi, and constitute the first demonstration of the presence and activation of this pathway in a parasitic protozoan. These results also indicate that this pathway is conserved during evolution from lower to higher eukaryotic organisms. Images Fig. 1. PMID:2025225

  13. Effects of ionizing radiation on struvite crystallization of livestock wastewater

    NASA Astrophysics Data System (ADS)

    Kim, Tak-Hyun; Nam, Yun-Ku; Joo Lim, Seung

    2014-04-01

    Livestock wastewater is generally very difficult to be treated by conventional wastewater treatment techniques because it contains high-strength organics (COD), ammonium (NH4+), phosphate (PO43-) and suspended solids. Struvite crystallization has been recently studied for the simultaneous removal of NH4+ and PO43-. In this study, gamma ray irradiation was carried out prior to struvite crystallization of the anaerobically digested livestock wastewater. The effects of gamma ray irradiation on the struvite crystallization of livestock wastewater were investigated. As a result, gamma ray irradiation can decrease the concentration of COD, NH4+ and PO43- contained in the livestock wastewater. This results in not only an enhancement of the struvite crystallization efficiency but also a decrease in the chemical demands for the struvite crystallization of livestock wastewater.

  14. Decoding the role of phosphoinositides in phototropin signaling involved in chloroplast movements.

    PubMed

    Aggarwal, Chhavi; Labuz, Justyna; Gabryś, Halina

    2013-08-01

    In angiosperms, light-dependent chloroplast movements are exclusively mediated by UVA/blue light receptors - phototropins. The two photoreceptors of Arabidopsis thaliana, phot1 and phot2, have overlapping roles in the control of these movements. Experiments performed in different plant species point to the participation of phosphoinositides in blue light-controlled chloroplast relocations. Here, we report a summary of recent findings presenting the involvement of phosphatidylinositol 4,5-bisphosphate as well as phosphatidylinositol 3- and 4-phosphates in weak blue light-mediated (accumulation) and strong blue light-mediated (avoidance) responses of chloroplasts. The blue light-activated alterations in phosphoinositide concentration are partly responsible for cytosolic Ca (2+) changes. Ca (2+) influx from apoplast does not seem to be involved in the mechanism of movement responses. In summary, interplay between phosphoinositides and intracellular Ca (2+) regulates chloroplast redistribution in response to blue light in higher plants.

  15. Interactions of legionella effector proteins with host phosphoinositide lipids.

    PubMed

    Weber, Stephen; Dolinsky, Stephanie; Hilbi, Hubert

    2013-01-01

    By means of the Icm/Dot type IV secretion system Legionella pneumophila translocates several effector proteins into host cells, where they anchor to the cytoplasmic face of the LCV membrane by binding to phosphoinositide (PI) lipids. Thus, phosphatidylinositol-4-phosphate anchors the effector proteins SidC and SidM, which promote the interaction of LCVs with the ER and the secretory vesicle trafficking -pathway. In this chapter, we describe protocols to (1) identify PI-binding proteins in Legionella lysates using PI-beads, (2) determine PI-binding specificities and affinities of recombinant Legionella effector proteins by protein-lipid overlays, and (3) use Legionella effectors to identify cellular PI lipids.

  16. Effect of transforming growth factor-alpha on inositol phospholipid metabolism in human epidermoid carcinoma cells

    SciTech Connect

    Kato, M.; Takenawa, T.; Twardzik, D.R.

    1988-08-01

    Transforming growth factor-alpha (TGF-alpha) stimulates (in a dose-dependent manner) the incorporation of (/sup 32/P)Pi into phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2), and phosphatidic acid (PA) in the human epidermoid carcinoma cell line (A431). The effect of TGF-alpha on the incorporation was found to be similar to that of EGF. On the other hand, a striking difference in the activation of diacylglycerol (DG) kinase activity was seen between TGF-alpha and EGF. At least 100 times more TGF-alpha was required to achieve maximal stimulation of DG kinase activity relative to EGF. These results suggest that the activation of DG kinase by TGF-alpha may involve a mechanism independent from or subsequent to activation of the EGF receptor.

  17. Expression and characterization of soluble 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase from bacterial pathogens.

    PubMed

    Eoh, Hyungjin; Narayanasamy, Prabagaran; Brown, Amanda C; Parish, Tanya; Brennan, Patrick J; Crick, Dean C

    2009-12-24

    Many bacterial pathogens utilize the 2-C-methyl-D-erythritol 4-phosphate pathway for biosynthesizing isoprenoid precursors, a pathway that is vital for bacterial survival and absent from human cells, providing a potential source of drug targets. However, the characterization of 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME) kinase (IspE) has been hindered due to a lack of enantiopure CDP-ME and difficulty in obtaining pure IspE. Here, enantiopure CDP-ME was chemically synthesized and recombinant IspE from bacterial pathogens were purified and characterized. Although gene disruption was not possible in Mycobacterium tuberculosis, IspE is essential in Mycobacterium smegmatis. The biochemical and kinetic characteristics of IspE provide the basis for development of a high throughput screen and structural characterization.

  18. FAM21 directs SNX27-retromer cargoes to the plasma membrane by preventing transport to the Golgi apparatus.

    PubMed

    Lee, Seongju; Chang, Jaerak; Blackstone, Craig

    2016-03-09

    The endosomal network maintains cellular homeostasis by sorting, recycling and degrading endocytosed cargoes. Retromer organizes the endosomal sorting pathway in conjunction with various sorting nexin (SNX) proteins. The SNX27-retromer complex has recently been identified as a major endosomal hub that regulates endosome-to-plasma membrane recycling by preventing lysosomal entry of cargoes. Here, we show that SNX27 directly interacts with FAM21, which also binds retromer, within the Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) complex. This interaction is required for the precise localization of SNX27 at an endosomal subdomain as well as for recycling of SNX27-retromer cargoes. Furthermore, FAM21 prevents cargo transport to the Golgi apparatus by controlling levels of phosphatidylinositol 4-phosphate, which facilitates cargo dissociation at the Golgi. Together, our results demonstrate that the SNX27-retromer-WASH complex directs cargoes to the plasma membrane by blocking their transport to lysosomes and the Golgi.

  19. Synthesis, sustained release properties of magnetically functionalized organic-inorganic materials: Amoxicillin anions intercalated magnetic layered double hydroxides via calcined precursors at room temperature

    NASA Astrophysics Data System (ADS)

    Wang, Jun; Liu, Qi; Zhang, Guangchun; Li, Zhanshuang; Yang, Piaoping; Jing, Xiaoyan; Zhang, Milin; Liu, Tianfu; Jiang, Zhaohua

    2009-09-01

    Zinc-aluminum-carbonate-layered double hydroxides (ZnAl-CO 3-LDHs), loaded with magnetic substrates (Fe 3O 4), were prepared for sustained drug-targeting delivery. From the X-ray diffraction results, it was found that the magnetic substrates were successfully incorporated with LDHs and highly dispersed in the hydrotalcite structure. After intercalation with an antibiotic drug (amoxicillin) by using a calcinations-reconstruction method, the basal spacing of layered double hydroxides increased from 7.51 Å to 12.35 Å, indicating that amoxicillin was successfully intercalated into the interlay space of LDHs as a monolayer. Furthermore, in vitro drug release experiments in pH 7.4 phosphate buffer solution (PBS) showed sustained release profiles with amoxicillin as a model drug. Magnetic measurements revealed that the composite possessed paramagnetic properties at room temperature.

  20. Effect of antidepressants and neuroleptics on phosphoinositide turnover in human platelets

    SciTech Connect

    Pandey, S.C.; Davis, J.M.; Schwertz, D.; Pandey, G.N. )

    1990-02-26

    The authors previously reported that tricyclic antidepressants and iprindole inhibit thrombin-stimulated formation of inositol-1, 4 bisphosphate (IP2) and inositol-1,4,5 triphosphate (IP3) but do not cause any change in inositol-1 phosphate (IP1). In order to examine if this decrease in IP2 and IP3 formation by antidepressants is related to the inhibition of the enzyme phospholipase C (PLC), the authors determined the effects of antidepressants and neuroleptics on the levels of 3(H) phosphotidylinositol (PI), 3(H) PI-4 phosphate (PIP), 3(H) PI-4, 5 bisphosphate (PIP2) in human platelets. The implications of the findings and their relevance to the mode of action of antidepressants are discussed.

  1. Antisense and chemical suppression of the nonmevalonate pathway affects ent-kaurene biosynthesis in Arabidopsis.

    PubMed

    Okada, Kazunori; Kawaide, Hiroshi; Kuzuyama, Tomohisa; Seto, Haruo; Curtis, Ian S; Kamiya, Yuji

    2002-06-01

    Transgenic plants of Arabidopsis thaliana (L.) Heynh. (ecotype Columbia) expressing the antisense AtMECT gene, encoding 2- C-methyl- D-erythritol 4-phosphate cytidylyltransferase, were generated to elucidate the physiological role of the nonmevalonate pathway for production of ent-kaurene, the latter being the plastidic precursor of gibberellins. In transformed plants pigmentation and accumulation of ent-kaurene were reduced compared to wild-type plants. Fosmidomycin, an inhibitor of 1-deoxy- D-xylulose 5-phosphate reductoisomerase (DXR), caused a similar depletion of these compounds in transgenic plants. These observations suggest that both AtMECT and DXR are important in the synthesis of isopentenyl diphosphate and dimethylallyl diphosphate and that ent-kaurene is mainly produced through the nonmevalonate pathway in the plastid.

  2. PI(4)P homeostasis: Who controls the controllers?

    PubMed

    Venditti, Rossella; Masone, Maria Chiara; Wilson, Cathal; De Matteis, Maria Antonietta

    2016-01-01

    During recent decades, PI(4)P (phosphoinositol-4-phosphate) has been described as a key regulator of a wide range of cellular functions such as organelle biogenesis, lipid metabolism and distribution, membrane trafficking, ion channels, pumps, and transporter activities. In this review we will focus on the multiple mechanisms that regulate PI(4)P homeostasis ranging from those responsible for the spatial distribution of the PI4 kinases and PI(4)P phosphatase to those controlling their enzymatic activity or the delivery/presentation of the substrate, i.e. PI or PI(4)P, to the PI4Ks or PI(4)P phosphatase, respectively. We will also highlight the open questions in the field mainly dealing with the existence and mode of action of PI(4)P sensors that monitor its amount and can act as a rheostat tuning PI(4)P levels in different compartments and adapting them to the different needs of the cell.

  3. Cloning, expression and characterization of sugarcane (Saccharum officinarum L.) transketolase.

    PubMed

    Kalhori, Nahid; Nulit, R; Go, Rusea

    2013-10-01

    Pentose phosphate pathway (PPP) composed of two functionally-connected phases, the oxidative and non-oxidative phase. Both phases catalysed by a series of enzymes. Transketolase is one of key enzymes of non-oxidative phase in which transfer two carbon units from fructose-6-phosphate to erythrose-4-phosphate and convert glyceraldehyde-3-phosphate to xylulose-5-phosphate. In plant, erythrose-4-phosphate enters the shikimate pathway which is produces many secondary metabolites such as aromatic amino acids, flavonoids, lignin. Although transketolase in plant system is important, study of this enzyme is still limited. Until to date, TKT genes had been isolated only from seven plants species, thus, the aim of present study to isolate, study the similarity and phylogeny of transketolase from sugarcane. Unlike bacteria, fungal and animal, PPP is complete in the cytosol and all enzymes are found cytosolic. However, in plant, the oxidative phase found localised in the cytosol but the sub localisation for non-oxidative phase might be restricted to plastid. Thus, this study was conducted to determine subcellular localization of sugarcane transketolase. The isolation of sugarcane TKT was done by reverse transcription polymerase chain reaction, followed by cloning into pJET1.2 vector and sequencing. This study has isolated 2,327 bp length of sugarcane TKT. The molecular phylogenetic tree analysis found that transketolase from sugarcane and Zea mays in one group. Classification analysis found that both plants showed closer relationship due to both plants in the same taxon i.e. family Poaceae. Target P 1.1 and Chloro P predicted that the compartmentation of sugarcane transketolase is localised in the chloroplast which is 85 amino acids are plant plastid target sequence. This led to conclusion that the PPP is incomplete in the cytosol of sugarcane. This study also found that the similarity sequence of sugarcane TKT closely related with the taxonomy plants.

  4. Contribution of granulocyte colony-stimulating factor to the acute mobilization of endothelial precursor cells by vascular disrupting agents.

    PubMed

    Shaked, Yuval; Tang, Terence; Woloszynek, Jill; Daenen, Laura G; Man, Shan; Xu, Ping; Cai, Shi-Rong; Arbeit, Jeffrey M; Voest, Emile E; Chaplin, David J; Smythe, Jon; Harris, Adrian; Nathan, Paul; Judson, Ian; Rustin, Gordon; Bertolini, Francesco; Link, Daniel C; Kerbel, Robert S

    2009-10-01

    Vascular disrupting agents (VDA) cause acute shutdown of abnormal established tumor vasculature, followed by massive intratumoral hypoxia and necrosis. However, a viable rim of tumor tissue invariably remains from which tumor regrowth rapidly resumes. We have recently shown that an acute systemic mobilization and homing of bone marrow-derived circulating endothelial precursor (CEP) cells could promote tumor regrowth following treatment with either a VDA or certain chemotherapy drugs. The molecular mediators of this systemic reactive host process are unknown. Here, we show that following treatment of mice with OXi-4503, a second-generation potent prodrug derivative of combretastatin-A4 phosphate, rapid increases in circulating plasma vascular endothelial growth factor, stromal derived factor-1 (SDF-1), and granulocyte colony-stimulating factor (G-CSF) levels are detected. With the aim of determining whether G-CSF is involved in VDA-induced CEP mobilization, mutant G-CSF-R(-/-) mice were treated with OXi-4503. We found that as opposed to wild-type controls, G-CSF-R(-/-) mice failed to mobilize CEPs or show induction of SDF-1 plasma levels. Furthermore, Lewis lung carcinomas grown in such mice treated with OXi-4503 showed greater levels of necrosis compared with tumors treated in wild-type mice. Evidence for rapid elevations in circulating plasma G-CSF, vascular endothelial growth factor, and SDF-1 were also observed in patients with VDA (combretastatin-A4 phosphate)-treated cancer. These results highlight the possible effect of drug-induced G-CSF on tumor regrowth following certain cytotoxic drug therapies, in this case using a VDA, and hence G-CSF as a possible therapeutic target.

  5. Biosynthesis of isoprenoids: a bifunctional IspDF enzyme from Campylobacter jejuni.

    PubMed

    Gabrielsen, Mads; Rohdich, Felix; Eisenreich, Wolfgang; Gräwert, Tobias; Hecht, Stefan; Bacher, Adelbert; Hunter, William N

    2004-07-01

    In the nonmevalonate pathway of isoprenoid biosynthesis, the conversion of 2C-methyl-d-erythritol 4-phosphate into its cyclic diphosphate proceeds via nucleotidyl intermediates and is catalyzed by the products of the ispD, ispE and ispF genes. An open reading frame of Campylobacter jejuni with similarity to the ispD and ispF genes of Escherichia coli was cloned into an expression vector directing the formation of a 42 kDa protein in a recombinant E. coli strain. The purified protein was shown to catalyze the transformation of 2C-methyl-D-erythritol 4-phosphate into 4-diphosphocytidyl-2C-methyl-D-erythritol and the conversion of 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate into 2C-methyl-D-erythritol 2,4-cyclodiphosphate at catalytic rates of 19 micro mol x mg(-1) x min(-1) and 7 micro mol x mg(-1) x min(-1), respectively. Both enzyme-catalyzed reactions require divalent metal ions. The C. jejuni enzyme does not catalyze the formation of 2C-methyl-D-erythritol 3,4-cyclophosphate from 4-diphosphocytidyl-2C-methyl-D-erythritol, a side reaction catalyzed in vitro by the IspF proteins of E. coli and Plasmodium falciparum. Comparative genomic analysis show that all sequenced alpha- and epsilon-proteobacteria have fused ispDF genes. These bifunctional proteins are potential drug targets in several human pathogens (e.g. Helicobacter pylori, C. jejuni and Treponema pallidum).

  6. Unravelling the regulatory mechanisms that modulate the MEP pathway in higher plants.

    PubMed

    Cordoba, Elizabeth; Salmi, Mari; León, Patricia

    2009-01-01

    The methyl-D-erythritol 4-phosphate pathway is responsible for the biosynthesis of a substantial number of natural compounds of biological and biotechnological importance. In recent years, this pathway has become an obvious target to develop new herbicides and antimicrobial drugs. In addition, the production of a variety of compounds of medical and agricultural interest may be possible through the genetic manipulation of this pathway. To this end, a complete understanding of the molecular mechanisms that regulate this pathway is of tremendous importance. Recent data have accumulated that show some of the multiple mechanisms that regulate the methyl-D-erythritol 4-phosphate pathway in plants. In this review we will describe some of these and discuss their implications. It has been demonstrated that 1-deoxy-D-xylulose-5-phosphate synthase (DXS), the first enzyme of this route, plays a major role in the overall regulation of the pathway. A small gene family codes for this enzyme in most of the plants which have been analysed so far, and the members of these gene families belong to different phylogenetic groups. Each of these genes exhibits a distinct expression pattern, suggesting unique functions. One of the most interesting regulatory mechanisms recently described for this pathway is the post-transcriptional regulation of the level of DXS and DXR proteins. In the case of DXS, this regulation appears conserved among plants, supporting its importance. The evidence accumulated suggests that this regulation might link the activity of this pathway with the plant's physiological conditions and the metabolic demand for the final products of this route.

  7. G protein-coupled receptor kinase GRK5 phosphorylates moesin and regulates metastasis in prostate cancer.

    PubMed

    Chakraborty, Prabir Kumar; Zhang, Yushan; Coomes, Alexandra S; Kim, Wan-Ju; Stupay, Rachel; Lynch, Lauren D; Atkinson, Tamieka; Kim, Jae I; Nie, Zhongzhen; Daaka, Yehia

    2014-07-01

    G protein-coupled receptor kinases (GRK) regulate diverse cellular functions ranging from metabolism to growth and locomotion. Here, we report an important contributory role for GRK5 in human prostate cancer. Inhibition of GRK5 kinase activity attenuated the migration and invasion of prostate cancer cells and, concordantly, increased cell attachment and focal adhesion formation. Mass spectrometric analysis of the phosphoproteome revealed the cytoskeletal-membrane attachment protein moesin as a putative GRK5 substrate. GRK5 regulated the subcellular distribution of moesin and colocalized with moesin at the cell periphery. We identified amino acid T66 of moesin as a principal GRK5 phosphorylation site and showed that enforcing the expression of a T66-mutated moesin reduced cell spreading. In a xenograft model of human prostate cancer, GRK5 silencing reduced tumor growth, invasion, and metastasis. Taken together, our results established GRK5 as a key contributor to the growth and metastasis of prostate cancer.

  8. Nerve Agent Exposure Elicits Site-Specific Changes in Protein Phosphorylation in Mouse Brain

    PubMed Central

    Zhu, Hongwen; O’Brien, Jennifer J.; O’Callaghan, James P.; Miller, Diane B.; Zhang, Qiang; Rana, Minal; Tsui, Tiffany; Peng, Youyi; Tomesch, John; Hendrick, Joseph P.; Wennogle, Lawrence P; Snyder, Gretchen L.

    2010-01-01

    Organophosphorus (OP) compounds cause toxic symptoms, including convulsions, coma, and death, as the result of irreversible inhibition of acetylcholinesterase (AChE). The development of effective treatments to block these effects and attenuate long-term cognitive and motor disabilities that result from OP intoxication is hampered by a limited understanding of the CNS pathways responsible for these actions. We employed a candidate method (called CNSProfile™) to identify changes in the phosphorylation state of key neuronal phosphoproteins evoked by the OP compound, diisopropyl fluorophosphate (DFP). Focused microwave fixation was used to preserve the phosphorylation state of phosphoproteins in brains of DFP-treated mice; hippocampus and striatum were analyzed by immunoblotting with a panel of phospho-specific antibodies. DFP exposure elicited comparable effects on phosphorylation of brain phosphoproteins in both C57BL/6 and FVB mice. DFP treatment significantly altered phosphorylation at regulatory residues on glutamate receptors, including Serine897 (S897) of the NR1 NMDA receptor. NR1 phosphorylation was bi-directionally regulated after DFP in striatum versus hippocampus. NR1 phosphorylation was reduced in striatum, but elevated in hippocampus, compared with controls. DARPP-32 phosphorylation in striatum was selectively increased at the Cdk5 kinase substrate, Threonine75 (T75). Phencynonate hydrochloride, a muscarinic cholinergic antagonist, prevented seizure-like behaviors and the observed changes in phosphorylation induced by DFP. The data reveal region-specific effects of nerve agent exposure on intracellular signaling pathways that correlate with seizure-like behavior and which are reversed by the muscarinic receptor blockade. This approach identifies specific targets for nerve agents, including substrates for Cdk5 kinase, which may be the basis for new anti-convulsant therapies. PMID:20423708

  9. The pathway for the production of inositol hexakisphosphate in human cells.

    PubMed

    Verbsky, John W; Chang, Shao-Chun; Wilson, Monita P; Mochizuki, Yasuhiro; Majerus, Philip W

    2005-01-21

    The yeast and Drosophila pathways leading to the production of inositol hexakisphosphate (InsP(6)) have been elucidated recently. The in vivo pathway in humans has been assumed to be similar. Here we show that overexpression of Ins(1,3,4)P(3) 5/6-kinase in human cell lines results in an increase of inositol tetrakisphosphate (InsP(4)) isomers, inositol pentakisphosphate (InsP(5)) and InsP(6), whereas its depletion by RNA interference decreases the amounts of these inositol phosphates. Expression of Ins(1,3,4,6)P(4) 5-kinase does not increase the amount of InsP(5) and InsP(6), although its depletion does block InsP(5) and InsP(6) production, showing that it is necessary for production of InsP(5) and InsP(6). Expression of Ins(1,3,4,5,6)P(5) 2-kinase increases the amount of InsP(6) by depleting the InsP(5) in the cell, and depletion of 2-kinase decreases the amount of InsP(6) and causes an increase in InsP(5). These results are consistent with a pathway that produces InsP(6) through the sequential action of Ins(1,3,4)P(3) 5/6-kinase, Ins(1,3,4,6)P(4) 5-kinase, and Ins(1,3,4,5,6)P5 2-kinase to convert Ins(1,3,4)P(3) to InsP(6). Furthermore, the evidence implicates 5/6-kinase as the rate-limiting enzyme in this pathway.

  10. Identification of the In Vivo Casein Kinase II Phosphorylation Site within the Homeodomain of the Cardiac Tisue-Specifying Homeobox Gene Product Csx/Nkx2.5

    PubMed Central

    Kasahara, Hideko; Izumo, Seigo

    1999-01-01

    Csx/Nkx2.5, a member of the homeodomain-containing transcription factors, serves critical developmental functions in heart formation in vertebrates and nonvertebrates. In this study the putative nuclear localization signal (NLS) of Csx/Nkx2.5 was identified by site-directed mutagenesis to the amino terminus of the homeodomain, which is conserved in almost all homeodomain proteins. When the putative NLS of Csx/Nkx2.5 was mutated a significant amount of the cytoplasmically localized Csx/Nkx2.5 was unphosphorylated, in contrast to the nuclearly localized Csx/Nkx2.5, which is serine- and threonine-phosphorylated, suggesting that Csx/Nkx2.5 phosphorylation is regulated, at least in part, by intracellular localization. Tryptic phosphopeptide mapping indicated that Csx/Nkx2.5 has at least five phosphorylation sites. Using in-gel kinase assays, we detected a Csx/Nkx2.5 kinase whose molecular mass is approximately 40 kDa in both cytoplasmic and nuclear extracts. Mutational analysis and in vitro kinase assays suggested that this 40-kDa Csx/Nkx2.5 kinase is a catalytic subunit of casein kinase II (CKII) that phosphorylates the serine residue between the first and second helix of the homeodomain. This CKII site is phosphorylated in vivo. CKII-dependent phosphorylation of the homeodomain increased Csx/Nkx2.5 DNA binding. Serine-to-alanine mutation at the CKII phosphorylation site reduced transcriptional activity when the carboxyl-terminal repressor domain was deleted. Although the precise biological function of Csx/Nkx2.5 phosphorylation by CKII remains to be determined, it may play an important role, as this CKII phosphorylation site within the homeodomain is fully conserved in all known members of the NK2 family of the homeobox genes. PMID:9858576

  11. Natural variation in monoterpene synthesis in kiwifruit: transcriptional regulation of terpene synthases by NAC and ETHYLENE-INSENSITIVE3-like transcription factors.

    PubMed

    Nieuwenhuizen, Niels J; Chen, Xiuyin; Wang, Mindy Y; Matich, Adam J; Perez, Ramon Lopez; Allan, Andrew C; Green, Sol A; Atkinson, Ross G

    2015-04-01

    Two kiwifruit (Actinidia) species with contrasting terpene profiles were compared to understand the regulation of fruit monoterpene production. High rates of terpinolene production in ripe Actinidia arguta fruit were correlated with increasing gene and protein expression of A. arguta terpene synthase1 (AaTPS1) and correlated with an increase in transcript levels of the 2-C-methyl-D-erythritol 4-phosphate pathway enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXS). Actinidia chinensis terpene synthase1 (AcTPS1) was identified as part of an array of eight tandemly duplicated genes, and AcTPS1 expression and terpene production were observed only at low levels in developing fruit. Transient overexpression of DXS in Nicotiana benthamiana leaves elevated monoterpene synthesis by AaTPS1 more than 100-fold, indicating that DXS is likely to be the key step in regulating 2-C-methyl-D-erythritol 4-phosphate substrate flux in kiwifruit. Comparative promoter analysis identified potential NAC (for no apical meristem [NAM], Arabidopsis transcription activation factor [ATAF], and cup-shaped cotyledon [CUC])-domain transcription factor) and ETHYLENE-INSENSITIVE3-like transcription factor (TF) binding sites in the AaTPS1 promoter, and cloned members of both TF classes were able to activate the AaTPS1 promoter in transient assays. Electrophoretic mobility shift assays showed that AaNAC2, AaNAC3, and AaNAC4 bind a 28-bp fragment of the proximal NAC binding site in the AaTPS1 promoter but not the A. chinensis AcTPS1 promoter, where the NAC binding site was mutated. Activation could be restored by reintroducing multiple repeats of the 12-bp NAC core-binding motif. The absence of NAC transcriptional activation in ripe A. chinensis fruit can account for the low accumulation of AcTPS1 transcript, protein, and monoterpene volatiles in this species. These results indicate the importance of NAC TFs in controlling monoterpene production and other traits in ripening fruits.

  12. Multiprobe Spectroscopic Inverstigation of Molecular-level Behavior within Aqueous 1-Butyl-3-methylimidazolium Tetrafluoroborate

    SciTech Connect

    Sarkar, Abhra; Ali, Maroof; Baker, Gary A; Tetin, Sergey Y.; Ruan, Qiaoqiao; Pandey, Siddharth

    2009-01-01

    In this work, an array of molecular-level solvent featuressincluding solute-solvent/solvent-solvent interactions, dipolarity, heterogeneity, dynamics, probe accessibility, and diffusionswere investigated across the entire composition of ambient mixtures containing the ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate, [bmim][BF4], and pH 7.0 phosphate buffer, based on results assembled for nine different molecular probes utilized in a range of spectroscopic modes. These studies uncovered interesting and unusual solvatochromic probe behavior within this benchmark mixture. Solvatochromic absorbance probessa watersoluble betaine dye (betaine dye 33), N,N-diethyl-4-nitroaniline, and 4-nitroanilineswere employed to determine ET (a blend of dipolarity/polarizability and hydrogen bond donor contributions) and the Kamlet-Taft indices * (dipolarity/polarizability), R (hydrogen bond donor acidity), and (hydrogen bond acceptor basicity) characterizing the [bmim][BF4] + phosphate buffer system. These parameters each showed a marked deviation from ideality, suggesting selective solvation of the individual probe solutes by [bmim][BF4]. Similar conclusions were derived from the responses of the fluorescent polarity-sensitive probes pyrene and pyrene-1-carboxaldehyde. Importantly, the fluorescent microfluidity probe 1,3-bis(1-pyrenyl)propane senses a microviscosity within the mixture that significantly exceeds expectations derived from simple interpolation of the behavior in the neat solvents. On the basis of results from this probe, a correlation between microviscosity and bulk viscosity was established; pronounced solvent-solvent hydrogen-bonding interactions were implicit in this behavior. The greatest deviation from ideal additive behavior for the probes studied herein was consistently observed to occur in the buffer-rich regime. Nitromethane-based fluorescence quenching of pyrene within the [bmim][BF4] + phosphate buffer system showed unusual compliance with a sphere

  13. Transaldolase B of Escherichia coli K-12: cloning of its gene, talB, and characterization of the enzyme from recombinant strains.

    PubMed Central

    Sprenger, G A; Schörken, U; Sprenger, G; Sahm, H

    1995-01-01

    A previously recognized open reading frame (T. Yura, H. Mori, H. Nagai, T. Nagata, A. Ishihama, N. Fujita, K. Isono, K. Mizobuchi, and A. Nakata, Nucleic Acids Res. 20:3305-3308) from the 0.2-min region of the Escherichia coli K-12 chromosome is shown to encode a functional transaldolase activity. After cloning of the gene onto high-copy-number vectors, transaldolase B (D-sedoheptulose-7-phosphate:D-glyceraldehyde-3-phosphate dihydroxyacetone transferase; EC 2.2.1.2) was overexpressed up to 12.7 U mg of protein-1 compared with less than 0.1 U mg of protein-1 in wild-type homogenates. The enzyme was purified from recombinant E. coli K-12 cells by successive ammonium sulfate precipitations (45 to 80% and subsequently 55 to 70%) and two anion-exchange chromatography steps (Q-Sepharose FF, Fractogel EMD-DEAE tentacle column; yield, 130 mg of protein from 12 g of cell wet weight) and afforded an apparently homogeneous protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a subunit size of 35,000 +/- 1,000 Da. As the enzyme had a molecular mass of 70,000 Da by gel filtration, transaldolase B is likely to form a homodimer. N-terminal amino acid sequencing of the protein verified its identity with the product of the cloned gene talB. The specific activity of the purified enzyme determined at 30 degrees C with the substrates fructose-6-phosphate (donor of C3 compound) and erythrose-4-phosphate (acceptor) at an optimal pH (50 mM glycylglycine [pH 8.5]) was 60 U mg-1.Km values for the substrates fructose-6-phosphate and erythrose-4-phosphate were determined at 1,200 and 90 microM, respectively. Kinetic constants for the other two physiological reactants, D,L-glyceraldehyde 3-phosphate (Km, 38 microM; relative activity [V(rel)], 8%) and sedoheptulose-7-phosphate (K(m), 285 microM; V(rel), 5%) were also determined. Fructose acted as a C(3) donor at a high apparent K(m) (>/=M) and with a V(rel) of 12%. The enzyme was inhibited by Tris-HCl, phosphate, or

  14. Natural Variation in Monoterpene Synthesis in Kiwifruit: Transcriptional Regulation of Terpene Synthases by NAC and ETHYLENE-INSENSITIVE3-Like Transcription Factors1

    PubMed Central

    Nieuwenhuizen, Niels J.; Chen, Xiuyin; Wang, Mindy Y.; Matich, Adam J.; Perez, Ramon Lopez; Allan, Andrew C.; Green, Sol A.; Atkinson, Ross G.

    2015-01-01

    Two kiwifruit (Actinidia) species with contrasting terpene profiles were compared to understand the regulation of fruit monoterpene production. High rates of terpinolene production in ripe Actinidia arguta fruit were correlated with increasing gene and protein expression of A. arguta terpene synthase1 (AaTPS1) and correlated with an increase in transcript levels of the 2-C-methyl-d-erythritol 4-phosphate pathway enzyme 1-deoxy-d-xylulose-5-phosphate synthase (DXS). Actinidia chinensis terpene synthase1 (AcTPS1) was identified as part of an array of eight tandemly duplicated genes, and AcTPS1 expression and terpene production were observed only at low levels in developing fruit. Transient overexpression of DXS in Nicotiana benthamiana leaves elevated monoterpene synthesis by AaTPS1 more than 100-fold, indicating that DXS is likely to be the key step in regulating 2-C-methyl-d-erythritol 4-phosphate substrate flux in kiwifruit. Comparative promoter analysis identified potential NAC (for no apical meristem [NAM], Arabidopsis transcription activation factor [ATAF], and cup-shaped cotyledon [CUC])-domain transcription factor) and ETHYLENE-INSENSITIVE3-like transcription factor (TF) binding sites in the AaTPS1 promoter, and cloned members of both TF classes were able to activate the AaTPS1 promoter in transient assays. Electrophoretic mobility shift assays showed that AaNAC2, AaNAC3, and AaNAC4 bind a 28-bp fragment of the proximal NAC binding site in the AaTPS1 promoter but not the A. chinensis AcTPS1 promoter, where the NAC binding site was mutated. Activation could be restored by reintroducing multiple repeats of the 12-bp NAC core-binding motif. The absence of NAC transcriptional activation in ripe A. chinensis fruit can account for the low accumulation of AcTPS1 transcript, protein, and monoterpene volatiles in this species. These results indicate the importance of NAC TFs in controlling monoterpene production and other traits in ripening fruits. PMID:25649633

  15. PtdIns4P recognition by Vps74/GOLPH3 links PtdIns 4-kinase signaling to retrograde Golgi trafficking

    SciTech Connect

    Wood, Christopher S.; Schmitz, Karl R.; Bessman, Nicholas J.; Setty, Thanuja Gangi; Ferguson, Kathryn M.; Burd, Christopher G.

    2010-02-11

    Targeting and retention of resident integral membrane proteins of the Golgi apparatus underly the function of the Golgi in glycoprotein and glycolipid processing and sorting. In yeast, steady-state Golgi localization of multiple mannosyltransferases requires recognition of their cytosolic domains by the peripheral Golgi membrane protein Vps74, an orthologue of human GOLPH3/GPP34/GMx33/MIDAS (mitochondrial DNA absence sensitive factor). We show that targeting of Vps74 and GOLPH3 to the Golgi apparatus requires ongoing synthesis of phosphatidylinositol (PtdIns) 4-phosphate (PtdIns4P) by the Pik1 PtdIns 4-kinase and that modulation of the levels and cellular location of PtdIns4P leads to mislocalization of these proteins. Vps74 and GOLPH3 bind specifically to PtdIns4P, and a sulfate ion in a crystal structure of GOLPH3 indicates a possible phosphoinositide-binding site that is conserved in Vps74. Alterations in this site abolish phosphoinositide binding in vitro and Vps74 function in vivo. These results implicate Pik1 signaling in retention of Golgi-resident proteins via Vps74 and show that GOLPH3 family proteins are effectors of Golgi PtdIns 4-kinases.

  16. Phenotypic Mutants of the Intracellular Actinomycete Rhodococcus equi Created by In Vivo Himar1 Transposon Mutagenesis

    PubMed Central

    Ashour, Joseph; Hondalus, Mary K.

    2003-01-01

    Rhodococcus equi is a facultative intracellular opportunistic pathogen of immunocompromised people and a major cause of pneumonia in young horses. An effective live attenuated vaccine would be extremely useful in the prevention of R. equi disease in horses. Toward that end, we have developed an efficient transposon mutagenesis system that makes use of a Himar1 minitransposon delivered by a conditionally replicating plasmid for construction of R. equi mutants. We show that Himar1 transposition in R. equi is random and needs no apparent consensus sequence beyond the required TA dinucleotide. The diversity of the transposon library was demonstrated by the ease with which we were able to screen for auxotrophs and mutants with pigmentation and capsular phenotypes. One of the pigmentation mutants contained an insertion in a gene encoding phytoene desaturase, an enzyme of carotenoid biosynthesis, the pathway necessary for production of the characteristic salmon color of R. equi. We identified an auxotrophic mutant with a transposon insertion in the gene encoding a putative dual-functioning GTP cyclohydrolase II-3,4-dihydroxy-2-butanone-4-phosphate synthase, an enzyme essential for riboflavin biosynthesis. This mutant cannot grow in minimal medium in the absence of riboflavin supplementation. Experimental murine infection studies showed that, in contrast to wild-type R. equi, the riboflavin-requiring mutant is attenuated because it is unable to replicate in vivo. The mutagenesis methodology we have developed will allow the characterization of R. equi virulence mechanisms and the creation of other attenuated strains with vaccine potential. PMID:12670990

  17. Up-regulation of connective tissue growth factor in endothelial cells by the microtubule-destabilizing agent combretastatin A-4.

    PubMed

    Samarin, Jana; Rehm, Margot; Krueger, Bettina; Waschke, Jens; Goppelt-Struebe, Margarete

    2009-02-01

    Incubation of microvascular endothelial cells with combretastatin A-4 phosphate (CA-4P), a microtubule-destabilizing compound that preferentially targets tumor vessels, altered cell morphology and induced scattering of Golgi stacks. Concomitantly, CA-4P up-regulated connective tissue growth factor (CTGF/CCN2), a pleiotropic factor with antiangiogenic properties. In contrast to the effects of other microtubule-targeting agents such as colchicine or nocodazole, up-regulation of CTGF was only detectable in sparse cells, which were not embedded in a cell monolayer. Furthermore, CA-4P induced CTGF expression in endothelial cells, forming tube-like structures on basement membrane gels. Up-regulation of CTGF by CA-4P was dependent on Rho kinase signaling and was increased when p42/44 mitogen-activated protein kinase was inhibited. Additionally, FoxO transcription factors were identified as potent regulators of CTGF expression in endothelial cells. Activation of FoxO transcription factors by inhibition of phosphatidylinositol 3-kinase/AKT signaling resulted in a synergistic increase in CA-4P-mediated CTGF induction. CA-4P-mediated expression of CTGF was thus potentiated by the inhibition of kinase pathways, which are targets of novel antineoplastic drugs. Up-regulation of CTGF by low concentrations of CA-4P may thus occur in newly formed tumor vessels and contribute to the microvessel destabilization and antiangiogenic effects of CA-4P observed in vivo.

  18. Structure of a Fbw7-Skp1-Cyclin E Complex: Multisite-Phosphorylated Substrate Recognition by SCF Ubiquitin Ligases

    SciTech Connect

    Hao,B.; Oehlmann, S.; Sowa, M.; Harper, J.; Pavletich, N.

    2007-01-01

    The ubiquitin-mediated proteolysis of cyclin E plays a central role in cell-cycle progression, and cyclin E accumulation is a common event in cancer. Cyclin E degradation is triggered by multisite phosphorylation, which induces binding to the SCFFbw7 ubiquitin ligase complex. Structures of the Skp1-Fbw7 complex bound to cyclin E peptides identify a doubly phosphorylated pThr380/pSer384 cyclin E motif as an optimal, high-affinity degron and a singly phosphorylated pThr62 motif as a low-affinity one. Biochemical data indicate that the closely related yeast SCFCdc4 complex recognizes the multisite phosphorylated Sic1 substrate similarly and identify three doubly phosphorylated Sic1 degrons, each capable of high-affinity interactions with two Cdc4 phosphate binding sites. A model that explains the role of multiple cyclin E/Sic1 degrons is provided by the findings that Fbw7 and Cdc4 dimerize, that Fbw7 dimerization enhances the turnover of a weakly associated cyclin E in vivo, and that Cdc4 dimerization increases the rate and processivity of Sic1 ubiquitination in vitro.

  19. Influence of phosphate ions on buffer capacity of soil humic acids

    NASA Astrophysics Data System (ADS)

    Boguta, P.; Sokołowska, Z.

    2012-02-01

    The object of this study was to determine change of natural buffer capacity of humic acids by strong buffering agents, which were phosphate ions. Studies were carried out on the humic acids extracted from peat soils. Additional information was obtained by determination of water holding capacity, density, ash and pH for peats and optical parameter Q4/6 for humic acids. Humic acid suspensions exhibited the highest buffer properties at low pH and reached maximum at pH ~ 4. Phosphates possessed buffer properties in the pH range from 4.5 to 8.0. The maximum of buffering was at pH~6.8 and increased proportionally with an increase in the concentration of phosphate ions. The study indicated that the presence of phosphate ions may strongly change natural buffer capacity of humic acids by shifting buffering maximum toward higher pH values. Significant correlations were found for the degree of the secondary transformation with both the buffer capacity and the titrant volume used during titration.

  20. Technical note: immunomagnetic procedure for positive selection of macrophages in ovine milk.

    PubMed

    Caroprese, M; Marzano, A; Schena, L; Sevi, A

    2008-05-01

    A simple immunomagnetic procedure was developed to select macrophages from ovine milk by using a non-specific magnetic positive separation technique. Samples of ewe bulk milk were collected during early, mid, and late lactation; milk samples were centrifuged at 2,000 x g for 30 min at 4 degrees C; the fatty fraction and supernatant were removed, and each pellet was dissolved in 500 microL of pH 7.4 phosphate buffered saline + 0.02% NaN(3). Cells were targeted for selection by using mouse-IgG anti-ovine macrophages. Several trials, testing 2 different fluorochrome-conjugated antibodies [i.e., mouse anti-human CD14:R-Phycoerythrin (RPE) (MCA1568PE, Serotec) and F(ab')2 rabbit anti-mouse IgG:RPE (STAR12A, Serotec)] and 3 different labeling procedures, were performed to evaluate the purity of samples by flow cytometry. A morphological test was carried out by direct microscopic count in enriched fraction smears stained with May-Grünwald-Giemsa stain to confirm the presence of macrophages. The method described in the present technical note can be considered an innovative application to obtain a single-cell population of high purity selected from all the somatic cells in milk.

  1. Enhanced accumulation of phytosterols and phenolic compounds in cyclodextrin-elicited cell suspension culture of Daucus carota.

    PubMed

    Miras-Moreno, Begoña; Almagro, Lorena; Pedreño, M A; Sabater-Jara, Ana Belén

    2016-09-01

    In this work, suspension-cultured cells of Daucus carota were used to evaluate the effect of β-cyclodextrins on the production of isoprenoid and phenolic compounds. The results showed that the phytosterols and phenolic compounds were accumulated in the extracellular medium (15100μgL(-1) and 477.46μgL(-1), respectively) in the presence of cyclodextrins. Unlike the phytosterol and phenolic compound content, β-carotene (1138.03μgL(-1)), lutein (25949.54μgL(-1)) and α-tocopherol (8063.82μgL(-1)) chlorophyll a (1625.13μgL(-1)) and b (9.958 (9958.33μgL(-1)) were mainly accumulated inside the cells. Therefore, cyclodextrins were able to induce the cytosolic mevalonate pathway, increasing the biosynthesis of phytosterols and phenolic compounds, and accumulate them outside the cells. However, in the absence of these cyclic oligosaccharidic elicitors, carrot cells mainly accumulated carotenoids through the methylerythritol 4-phosphate pathway. Therefore, the use of cyclodextrins would allow the extracellular accumulation of both phytosterols and phenolic compounds by diverting the carbon flux towards the cytosolic mevalonate/phenylpropanoid pathway.

  2. The N-Myc down regulated Gene1 (NDRG1) Is a Rab4a effector involved in vesicular recycling of E-cadherin.

    PubMed

    Kachhap, Sushant K; Faith, Dennis; Qian, David Z; Shabbeer, Shabana; Galloway, Nathan L; Pili, Roberto; Denmeade, Samuel R; DeMarzo, Angelo M; Carducci, Michael A

    2007-09-05

    Cell to cell adhesion is mediated by adhesion molecules present on the cell surface. Downregulation of molecules that form the adhesion complex is a characteristic of metastatic cancer cells. Downregulation of the N-myc down regulated gene1 (NDRG1) increases prostate and breast metastasis. The exact function of NDRG1 is not known. Here by using live cell confocal microscopy and in vitro reconstitution, we report that NDRG1 is involved in recycling the adhesion molecule E-cadherin thereby stabilizing it. Evidence is provided that NDRG1 recruits on recycling endosomes in the Trans Golgi network by binding to phosphotidylinositol 4-phosphate and interacts with membrane bound Rab4aGTPase. NDRG1 specifically interacts with constitutively active Rab4aQ67L mutant protein and not with GDP-bound Rab4aS22N mutant proving NDRG1 as a novel Rab4a effector. Transferrin recycling experiments reveals NDRG1 colocalizes with transferrin during the recycling phase. NDRG1 alters the kinetics of transferrin recycling in cells. NDRG1 knockdown cells show a delay in recycling transferrin, conversely NDRG1 overexpressing cells reveal an increase in rate of transferrin recycling. This novel finding of NDRG1 as a recycling protein involved with recycling of E-cadherin will aid in understanding NDRG1 role as a metastasis suppressor protein.

  3. Bisubstrate analogue inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase: New design with improved properties

    SciTech Connect

    Shi, Genbin; Shaw, Gary; Liang, Yu-He; Subburaman, Priadarsini; Li, Yue; Wu, Yan; Yan, Honggao; Ji, Xinhua

    2012-07-11

    6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), a key enzyme in the folate biosynthetic pathway, catalyzes the pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin. The enzyme is essential for microorganisms, is absent from humans, and is not the target for any existing antibiotics. Therefore, HPPK is an attractive target for developing novel antimicrobial agents. Previously, we characterized the reaction trajectory of HPPK-catalyzed pyrophosphoryl transfer and synthesized a series of bisubstrate analog inhibitors of the enzyme by linking 6-hydroxymethylpterin to adenosine through 2, 3, or 4 phosphate groups. Here, we report a new generation of bisubstrate analog inhibitors. To improve protein binding and linker properties of such inhibitors, we have replaced the pterin moiety with 7,7-dimethyl-7,8-dihydropterin and the phosphate bridge with a piperidine linked thioether. We have synthesized the new inhibitors, measured their K{sub d} and IC{sub 50} values, determined their crystal structures in complex with HPPK, and established their structure-activity relationship. 6-Carboxylic acid ethyl ester-7,7-dimethyl-7,8-dihydropterin, a novel intermediate that we developed recently for easy derivatization at position 6 of 7,7-dimethyl-7,8-dihydropterin, offers a much high yield for the synthesis of bisubstrate analogs than that of previously established procedure.

  4. Tuning a cellular lipid kinase activity adapts hepatitis C virus to replication in cell culture.

    PubMed

    Harak, Christian; Meyrath, Max; Romero-Brey, Inés; Schenk, Christian; Gondeau, Claire; Schult, Philipp; Esser-Nobis, Katharina; Saeed, Mohsan; Neddermann, Petra; Schnitzler, Paul; Gotthardt, Daniel; Perez-Del-Pulgar, Sofia; Neumann-Haefelin, Christoph; Thimme, Robert; Meuleman, Philip; Vondran, Florian W R; Francesco, Raffaele De; Rice, Charles M; Bartenschlager, Ralf; Lohmann, Volker

    2016-12-19

    With a single exception, all isolates of hepatitis C virus (HCV) require adaptive mutations to replicate efficiently in cell culture. Here, we show that a major class of adaptive mutations regulates the activity of a cellular lipid kinase, phosphatidylinositol 4-kinase IIIα (PI4KA). HCV needs to stimulate PI4KA to create a permissive phosphatidylinositol 4-phosphate-enriched membrane microenvironment in the liver and in primary human hepatocytes (PHHs). In contrast, in Huh7 hepatoma cells, the virus must acquire loss-of-function mutations that prevent PI4KA overactivation. This adaptive mechanism is necessitated by increased PI4KA levels in Huh7 cells compared with PHHs, and is conserved across HCV genotypes. PI4KA-specific inhibitors promote replication of unadapted viral isolates and allow efficient replication of patient-derived virus in cell culture. In summary, this study has uncovered a long-sought mechanism of HCV cell-culture adaptation and demonstrates how a virus can adapt to changes in a cellular environment associated with tumorigenesis.

  5. Improving peppermint essential oil yield and composition by metabolic engineering.

    PubMed

    Lange, Bernd Markus; Mahmoud, Soheil Seyed; Wildung, Mark R; Turner, Glenn W; Davis, Edward M; Lange, Iris; Baker, Raymond C; Boydston, Rick A; Croteau, Rodney B

    2011-10-11

    Peppermint (Mentha × piperita L.) was transformed with various gene constructs to evaluate the utility of metabolic engineering for improving essential oil yield and composition. Oil yield increases were achieved by overexpressing genes involved in the supply of precursors through the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. Two-gene combinations to enhance both oil yield and composition in a single transgenic line were assessed as well. The most promising results were obtained by transforming plants expressing an antisense version of (+)-menthofuran synthase, which is critical for adjusting the levels of specific undesirable oil constituents, with a construct for the overexpression of the MEP pathway gene 1-deoxy-D-xylulose 5-phosphate reductoisomerase (up to 61% oil yield increase over wild-type controls with low levels of the undesirable side-product (+)-menthofuran and its intermediate (+)-pulegone). Elite transgenic lines were advanced to multiyear field trials, which demonstrated consistent oil yield increases of up to 78% over wild-type controls and desirable effects on oil composition under commercial growth conditions. The transgenic expression of a gene encoding (+)-limonene synthase was used to accumulate elevated levels of (+)-limonene, which allows oil derived from transgenic plants to be recognized during the processing of commercial formulations containing peppermint oil. Our study illustrates the utility of metabolic engineering for the sustainable agricultural production of high quality essential oils at a competitive cost.

  6. Carotenoid Derivates in Achiote (Bixa orellana) Seeds: Synthesis and Health Promoting Properties.

    PubMed

    Rivera-Madrid, Renata; Aguilar-Espinosa, Margarita; Cárdenas-Conejo, Yair; Garza-Caligaris, Luz E

    2016-01-01

    Bixa orellana (family Bixaceae) is a neotropical fast growing perennial tree of great agro-industrial value because its seeds have a high carotenoid content, mainly bixin. It has been used since pre-colonial times as a culinary colorant and spice, and for healing purposes. It is currently used as a natural pigment in the food, in pharmaceutical, and cosmetic industries, and it is commercially known as annatto. Recently, several studies have addressed the biological and medical properties of this natural pigment, both as potential source of new drugs or because its ingestion as a condiment or diet supplement may protect against several diseases. The most documented properties are anti-oxidative; but its anti-cancer, hypoglucemic, antibiotic and anti-inflammatory properties are also being studied. Bixin's pathway elucidation and its regulation mechanisms are critical to improve the produce of this important carotenoid. Even though the bixin pathway has been established, the regulation of the genes involved in bixin production remains largely unknown. Our laboratory recently published B. orellana's transcriptome and we have identified most of its MEP (methyl-D-erythritol 4-phosphate) and carotenoid pathway genes. Annatto is a potential source of new drugs and can be a valuable nutraceutical supplement. However, its nutritional and healing properties require further study.

  7. Riboflavin synthesis genes are linked with the lux operon of Photobacterium phosphoreum.

    PubMed Central

    Lee, C Y; O'Kane, D J; Meighen, E A

    1994-01-01

    Four genes immediately downstream of luxG in the Photobacterium phosphoreum lux operon (ribEBHA) have been sequenced and shown to be involved in riboflavin synthesis. Sequence analyses and complementation of Escherichia coli riboflavin auxotrophs showed that the gene products of ribB and ribA are 3,4-dihydroxy-2-butanone 4-phosphate (DHBP) synthetase and GTP cyclohydrolase II, respectively. By expression of P. phosphoreum ribE in E. coli using the bacteriophage T7 promoter-RNA polymerase system, ribE was shown to code for riboflavin synthetase, which catalyzes the conversion of lumazine to riboflavin. Increased thermal stability of RibE on expression with RibH indicated that ribH coded for lumazine synthetase. The organization of the rib genes in P. phosphoreum is quite distinct, with ribB and ribA being linked but separated by ribH, whereas in E. coli, they are unlinked and in Bacillus subtilis, RibB and RibA functions are coded by a single gene. Images PMID:8144477

  8. Identifying novel genes and chemicals related to nasopharyngeal cancer in a heterogeneous network

    PubMed Central

    Li, Zhandong; An, Lifeng; Li, Hao; Wang, ShaoPeng; Zhou, You; Yuan, Fei; Li, Lin

    2016-01-01

    Nasopharyngeal cancer or nasopharyngeal carcinoma (NPC) is the most common cancer originating in the nasopharynx. The factors that induce nasopharyngeal cancer are still not clear. Additional information about the chemicals or genes related to nasopharyngeal cancer will promote a better understanding of the pathogenesis of this cancer and the factors that induce it. Thus, a computational method NPC-RGCP was proposed in this study to identify the possible relevant chemicals and genes based on the presently known chemicals and genes related to nasopharyngeal cancer. To extensively utilize the functional associations between proteins and chemicals, a heterogeneous network was constructed based on interactions of proteins and chemicals. The NPC-RGCP included two stages: the searching stage and the screening stage. The former stage is for finding new possible genes and chemicals in the heterogeneous network, while the latter stage is for screening and removing false discoveries and selecting the core genes and chemicals. As a result, five putative genes, CXCR3, IRF1, CDK1, GSTP1, and CDH2, and seven putative chemicals, iron, propionic acid, dimethyl sulfoxide, isopropanol, erythrose 4-phosphate, β-D-Fructose 6-phosphate, and flavin adenine dinucleotide, were identified by NPC-RGCP. Extensive analyses provided confirmation that the putative genes and chemicals have significant associations with nasopharyngeal cancer. PMID:27149165

  9. Environmental and Genetic Factors Associated with Solanesol Accumulation in Potato Leaves

    PubMed Central

    Campbell, Raymond; Freitag, Sabine; Bryan, Glenn J.; Stewart, Derek; Taylor, Mark A.

    2016-01-01

    Solanesol is a high value 45-carbon, unsaturated, all-trans-nonaprenol isoprenoid. Recently solanesol has received particular attention because of its utility, both in its own right and as a precursor in the production of numerous compounds used in the treatment of disease states. Solanesol is found mainly in solanaceous crops such as potato, tomato, tobacco and pepper where it accumulates in the foliage. There is considerable potential to explore the extraction of solanesol from these sources as a valuable co-product. In this study we have characterized the genetic variation in leaf solanesol content in a biparental, segregating diploid potato population. We demonstrate that potato leaf solanesol content is genetically controlled and identify several quantitative trait loci associated with leaf solanesol content. Transient over-expression of genes from the methylerythritol 4-phosphate (MEP) and mevalonic acid (MVA) pathways, either singly or in combination, resulted in enhanced accumulation of solanesol in leaves of Nicotiana benthamiana, providing insights for genetically engineering the pathway. We also demonstrate that in potato, leaf solanesol content is enhanced by up to six-fold on exposure to moderately elevated temperature and show corresponding changes in expression patterns of MEP and MVA genes. Our combined approaches offer new insights into solanesol accumulation and strategies for developing a bio-refinery approach to potato production. PMID:27610114

  10. Cloning and Expression Analysis of MEP Pathway Enzyme-encoding Genes in Osmanthus fragrans

    PubMed Central

    Xu, Chen; Li, Huogeng; Yang, Xiulian; Gu, Chunsun; Mu, Hongna; Yue, Yuanzheng; Wang, Lianggui

    2016-01-01

    The 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway is responsible for the biosynthesis of many crucial secondary metabolites, such as carotenoids, monoterpenes, plastoquinone, and tocopherols. In this study, we isolated and identified 10 MEP pathway genes in the important aromatic plant sweet osmanthus (Osmanthus fragrans). Multiple sequence alignments revealed that 10 MEP pathway genes shared high identities with other reported proteins. The genes showed distinctive expression profiles in various tissues, or at different flower stages and diel time points. The qRT-PCR results demonstrated that these genes were highly expressed in inflorescences, which suggested a tissue-specific transcript pattern. Our results also showed that OfDXS1, OfDXS2, and OfHDR1 had a clear diurnal oscillation pattern. The isolation and expression analysis provides a strong foundation for further research on the MEP pathway involved in gene function and molecular evolution, and improves our understanding of the molecular mechanism underlying this pathway in plants. PMID:27690108

  11. Interaction of FAPP1 with ARF1 and PI4P at a membrane surface: an example of coincidence detection

    PubMed Central

    Liu, Yizhou; Kahn, Richard A.; Prestegard, James H.

    2014-01-01

    SUMMARY Interactions among ADP-ribosylation factors (ARFs), various adaptor proteins, and membrane lipids are essential for intracellular vesicle transport of a variety of cellular materials. Here we present NMR-based information on the nature of the interaction of yeast ARF1 (yARF1) and the pleckstrin homology (PH) domain of four-phosphate-adaptor protein 1 (FAPP1) as it occurs at a model membrane surface. Interactions favor a model in which FAPP1 is partially embedded in the membrane and interacts with a membrane-associated ARF1 molecule primarily through contacts between residues in switch I of ARF1 and regions near and under the solution exposed C-terminal extension of the PH domain. The ARF1 binding site on FAPP1-PH is distinct from a positively charged phosphatidylinositol-4-phosphate (PI4P) binding site. A structural model is constructed that supports coincidence detection of both activated ARF and PI4P as a mechanism facilitating FAPP1 recruitment to membranes. PMID:24462251

  12. Synthetic Routes to Methylerythritol Phosphate Pathway Intermediates and Downstream Isoprenoids

    PubMed Central

    Jarchow-Choy, Sarah K; Koppisch, Andrew T; Fox, David T

    2014-01-01

    Isoprenoids constitute the largest class of natural products with greater than 55,000 identified members. They play essential roles in maintaining proper cellular function leading to maintenance of human health, plant defense mechanisms against predators, and are often exploited for their beneficial properties in the pharmaceutical and nutraceutical industries. Most impressively, all known isoprenoids are derived from one of two C5-precursors, isopentenyl diphosphate (IPP) or dimethylallyl diphosphate (DMAPP). In order to study the enzyme transformations leading to the extensive structural diversity found within this class of compounds there must be access to the substrates. Sometimes, intermediates within a biological pathway can be isolated and used directly to study enzyme/pathway function. However, the primary route to most of the isoprenoid intermediates is through chemical catalysis. As such, this review provides the first exhaustive examination of synthetic routes to isoprenoid and isoprenoid precursors with particular emphasis on the syntheses of intermediates found as part of the 2C-methylerythritol 4-phosphate (MEP) pathway. In addition, representative syntheses are presented for the monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), triterpenes (C30) and tetraterpenes (C40). Finally, in some instances, the synthetic routes to substrate analogs found both within the MEP pathway and downstream isoprenoids are examined. PMID:25009443

  13. Regulation of Primary Metabolic Pathways in Oyster Mushroom Mycelia Induced by Blue Light Stimulation: Accumulation of Shikimic Acid

    PubMed Central

    Kojima, Masanobu; Kimura, Ninako; Miura, Ryuhei

    2015-01-01

    Shikimic acid is a key intermediate in the aromatic amino acid pathway as well as an important starting material for the synthesis of Tamiflu, a potent and selective inhibitor of the neuraminidase enzyme of influenza viruses A and B. Here we report that in oyster mushroom (Pleurotus ostreatus) mycelia cultivated in the dark, stimulation with blue light-emitting diodes induces the accumulation of shikimic acid. An integrated analysis of primary metabolites, gene expression and protein expression suggests that the accumulation of shikimic acid caused by blue light stimulation is due to an increase in 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (DAHPS, EC2.5.1.54), the rate-determining enzyme in the shikimic acid pathway, as well as phosphofructokinase (PFK, EC2.7.1.11) and glucose-6-phosphate dehydrogenase (G6PD, EC1.1.1.49), the rate-determining enzymes in the glycolysis and pentose phosphate pathways, respectively. This stimulation results in increased levels of phosphoenolpyruvic acid (PEP) and erythrose-4-phosphate (E4P), the starting materials of shikimic acid biosynthesis. PMID:25721093

  14. Enhanced Diterpene Tanshinone Accumulation and Bioactivity of Transgenic Salvia miltiorrhiza Hairy Roots by Pathway Engineering.

    PubMed

    Shi, Min; Luo, Xiuqin; Ju, Guanhua; Li, Leilei; Huang, Shengxiong; Zhang, Tong; Wang, Huizhong; Kai, Guoyin

    2016-03-30

    Tanshinones are health-promoting diterpenoids found in Salvia miltiorrhiza and have wide applications. Here, SmGGPPS (geranylgeranyl diphosphate synthase) and SmDXSII (1-deoxy-D-xylulose-5-phosphate synthase) were introduced into hairy roots of S. miltiorrhiza. Overexpression of SmGGPPS and SmDXSII in hairy roots produces higher levels of tanshinone than control and single-gene transformed lines; tanshinone production in the double-gene transformed line GDII10 reached 12.93 mg/g dry weight, which is the highest tanshinone content that has been achieved through genetic engineering. Furthermore, transgenic hairy root lines showed higher antioxidant and antitumor activities than control lines. In addition, contents of chlorophylls, carotenoids, indoleacetic acid, and gibberellins were significantly elevated in transgenic Arabidopsis thaliana plants. These results demonstrate a promising method to improve the production of diterpenoids including tanshinone as well as other natural plastid-derived isoprenoids in plants by genetic manipulation of the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway.

  15. Norepinephrine and veratrine stimulated formation of inositol phosphates in rat brain slices

    SciTech Connect

    Maier, K.U.; Rutledge, C.O.

    1986-03-05

    Stimulation of phosphoinositide (PIn) hydrolysis by depolarization with veratrine was compared to that produced by stimulation of alpha/sub 1/ adrenoceptors by norepinephrine (NE). The PIns in rat cerebral cortex were labelled with /sup 3/H-myoinositol and the effects of the drugs on the formation of the three /sup 3/H-inositol phosphates (IP, IP2, IP3) were determined. The amounts of IP and IP2 formed by a maximal concentration of veratrine were about 50% of that formed by a maximal concentration of NE while the amount of IP3 formed after stimulation by veratrine was only about 10% of that produced by NE. The increase in IP was linear with time (30 min) for both NE and veratrine. IP2 and IP3 stimulation by veratrine reached a maximum at 5 min whereas that produced by NE continued to increase for 30 min. Blockade of voltage dependent calcium channels with manganese produced nearly complete antagonism of the veratrine response while only partially antagonizing the NE response. NE-induced IP2 formation was less sensitive to manganese than IP or IP3. These data suggest that veratrine causes hydrolysis of either a different pool of PIn or that the hydrolysis occurs by a different mechanism compared to NE. The data also suggest that IP2 may be produced directly from phosphatidylinositol 4-phosphate rather than solely as a metabolite of IP3.

  16. Type II PI4-kinases control Weibel-Palade body biogenesis and von Willebrand factor structure in human endothelial cells

    PubMed Central

    Lopes da Silva, Mafalda; O'Connor, Marie N.; Kriston-Vizi, Janos; White, Ian J.; Al-Shawi, Raya; Simons, J. Paul; Mössinger, Julia; Haucke, Volker

    2016-01-01

    ABSTRACT Weibel-Palade bodies (WPBs) are endothelial storage organelles that mediate the release of molecules involved in thrombosis, inflammation and angiogenesis, including the pro-thrombotic glycoprotein von Willebrand factor (VWF). Although many protein components required for WPB formation and function have been identified, the role of lipids is almost unknown. We examined two key phosphatidylinositol kinases that control phosphatidylinositol 4-phosphate levels at the trans-Golgi network, the site of WPB biogenesis. RNA interference of the type II phosphatidylinositol 4-kinases PI4KIIα and PI4KIIβ in primary human endothelial cells leads to formation of an increased proportion of short WPB with perturbed packing of VWF, as exemplified by increased exposure of antibody-binding sites. When stimulated with histamine, these cells release normal levels of VWF yet, under flow, form very few platelet-catching VWF strings. In PI4KIIα-deficient mice, immuno-microscopy revealed that VWF packaging is also perturbed and these mice exhibit increased blood loss after tail cut compared to controls. This is the first demonstration that lipid kinases can control the biosynthesis of VWF and the formation of WPBs that are capable of full haemostatic function. PMID:27068535

  17. Endosomal sorting of VAMP3 is regulated by PI4K2A.

    PubMed

    Jović, Marko; Kean, Michelle J; Dubankova, Anna; Boura, Evzen; Gingras, Anne-Claude; Brill, Julie A; Balla, Tamas

    2014-09-01

    Specificity of membrane fusion in vesicular trafficking is dependent on proper subcellular distribution of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). Although SNARE complexes are fairly promiscuous in vitro, substantial specificity is achieved in cells owing to the spatial segregation and shielding of SNARE motifs prior to association with cognate Q-SNAREs. In this study, we identified phosphatidylinositol 4-kinase IIα (PI4K2A) as a binding partner of vesicle-associated membrane protein 3 (VAMP3), a small R-SNARE involved in recycling and retrograde transport, and found that the two proteins co-reside on tubulo-vesicular endosomes. PI4K2A knockdown inhibited VAMP3 trafficking to perinuclear membranes and impaired the rate of VAMP3-mediated recycling of the transferrin receptor. Moreover, depletion of PI4K2A significantly decreased association of VAMP3 with its cognate Q-SNARE Vti1a. Although binding of VAMP3 to PI4K2A did not require kinase activity, acute depletion of phosphatidylinositol 4-phosphate (PtdIns4P) on endosomes significantly delayed VAMP3 trafficking. Modulation of SNARE function by phospholipids had previously been proposed based on in vitro studies, and our study provides mechanistic evidence in support of these claims by identifying PI4K2A and PtdIns4P as regulators of an R-SNARE in intact cells.

  18. Targeting Plasmodium PI(4)K to eliminate malaria

    NASA Astrophysics Data System (ADS)

    McNamara, Case W.; Lee, Marcus C. S.; Lim, Chek Shik; Lim, Siau Hoi; Roland, Jason; Nagle, Advait; Simon, Oliver; Yeung, Bryan K. S.; Chatterjee, Arnab K.; McCormack, Susan L.; Manary, Micah J.; Zeeman, Anne-Marie; Dechering, Koen J.; Kumar, T. R. Santha; Henrich, Philipp P.; Gagaring, Kerstin; Ibanez, Maureen; Kato, Nobutaka; Kuhen, Kelli L.; Fischli, Christoph; Rottmann, Matthias; Plouffe, David M.; Bursulaya, Badry; Meister, Stephan; Rameh, Lucia; Trappe, Joerg; Haasen, Dorothea; Timmerman, Martijn; Sauerwein, Robert W.; Suwanarusk, Rossarin; Russell, Bruce; Renia, Laurent; Nosten, Francois; Tully, David C.; Kocken, Clemens H. M.; Glynne, Richard J.; Bodenreider, Christophe; Fidock, David A.; Diagana, Thierry T.; Winzeler, Elizabeth A.

    2013-12-01

    Achieving the goal of malaria elimination will depend on targeting Plasmodium pathways essential across all life stages. Here we identify a lipid kinase, phosphatidylinositol-4-OH kinase (PI(4)K), as the target of imidazopyrazines, a new antimalarial compound class that inhibits the intracellular development of multiple Plasmodium species at each stage of infection in the vertebrate host. Imidazopyrazines demonstrate potent preventive, therapeutic, and transmission-blocking activity in rodent malaria models, are active against blood-stage field isolates of the major human pathogens P. falciparum and P. vivax, and inhibit liver-stage hypnozoites in the simian parasite P. cynomolgi. We show that imidazopyrazines exert their effect through inhibitory interaction with the ATP-binding pocket of PI(4)K, altering the intracellular distribution of phosphatidylinositol-4-phosphate. Collectively, our data define PI(4)K as a key Plasmodium vulnerability, opening up new avenues of target-based discovery to identify drugs with an ideal activity profile for the prevention, treatment and elimination of malaria.

  19. Nanoscale Landscape of Phosphoinositides Revealed by Specific Pleckstrin Homology (PH) Domains Using Single-molecule Superresolution Imaging in the Plasma Membrane*

    PubMed Central

    Ji, Chen; Zhang, Yongdeng; Xu, Pingyong; Xu, Tao; Lou, Xuelin

    2015-01-01

    Both phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) are independent plasma membrane (PM) determinant lipids that are essential for multiple cellular functions. However, their nanoscale spatial organization in the PM remains elusive. Using single-molecule superresolution microscopy and new photoactivatable fluorescence probes on the basis of pleckstrin homology domains that specifically recognize phosphatidylinositides in insulin-secreting INS-1 cells, we report that the PI(4,5)P2 probes exhibited a remarkably uniform distribution in the major regions of the PM, with some sparse PI(4,5)P2-enriched membrane patches/domains of diverse sizes (383 ± 14 nm on average). Quantitative analysis revealed a modest concentration gradient that was much less steep than previously thought, and no densely packed PI(4,5)P2 nanodomains were observed. Live-cell superresolution imaging further demonstrated the dynamic structural changes of those domains in the flat PM and membrane protrusions. PI4P and phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) showed similar spatial distributions as PI(4,5)P2. These data reveal the nanoscale landscape of key inositol phospholipids in the native PM and imply a framework for local cellular signaling and lipid-protein interactions at a nanometer scale. PMID:26396197

  20. PI(4)P Promotes Phosphorylation and Conformational Change of Smoothened through Interaction with Its C-terminal Tail

    PubMed Central

    Zhang, Jie; Li, Xiang-An; Evers, B. Mark; Zhu, Haining; Jia, Jianhang

    2016-01-01

    In Hedgehog (Hh) signaling, binding of Hh to the Patched-Interference Hh (Ptc-Ihog) receptor complex relieves Ptc inhibition on Smoothened (Smo). A longstanding question is how Ptc inhibits Smo and how such inhibition is relieved by Hh stimulation. In this study, we found that Hh elevates production of phosphatidylinositol 4-phosphate (PI(4)P). Increased levels of PI(4)P promote, whereas decreased levels of PI(4)P inhibit, Hh signaling activity. We further found that PI(4)P directly binds Smo through an arginine motif, which then triggers Smo phosphorylation and activation. Moreover, we identified the pleckstrin homology (PH) domain of G protein-coupled receptor kinase 2 (Gprk2) as an essential component for enriching PI(4)P and facilitating Smo activation. PI(4)P also binds mouse Smo (mSmo) and promotes its phosphorylation and ciliary accumulation. Finally, Hh treatment increases the interaction between Smo and PI(4)P but decreases the interaction between Ptc and PI(4)P, indicating that, in addition to promoting PI(4)P production, Hh regulates the pool of PI(4)P associated with Ptc and Smo. PMID:26863604

  1. Structural elements that govern Sec14-like PITP sensitivities to potent small molecule inhibitors.

    PubMed

    Khan, Danish; McGrath, Kaitlyn R; Dorosheva, Oleksandra; Bankaitis, Vytas A; Tripathi, Ashutosh

    2016-04-01

    Sec14-like phosphatidylinositol transfer proteins (PITPs) play important biological functions in integrating multiple aspects of intracellular lipid metabolism with phosphatidylinositol-4-phosphate signaling. As such, these proteins offer new opportunities for highly selective chemical interference with specific phosphoinositide pathways in cells. The first and best characterized small molecule inhibitors of the yeast PITP, Sec14, are nitrophenyl(4-(2-methoxyphenyl)piperazin-1-yl)methanones (NPPMs), and a hallmark feature of NPPMs is their exquisite targeting specificities for Sec14 relative to other closely related Sec14-like PITPs. Our present understanding of Sec14::NPPM binding interactions is based on computational docking and rational loss-of-function approaches. While those approaches have been informative, we still lack an adequate understanding of the basis for the high selectivity of NPPMs among closely related Sec14-like PITPs. Herein, we describe a Sec14 motif, which we term the VV signature, that contributes significantly to the NPPM sensitivity/resistance of Sec14-like phosphatidylinositol (PtdIns)/phosphatidylcholine (PtdCho) transfer proteins. The data not only reveal previously unappreciated determinants that govern Sec14-like PITP sensitivities to NPPMs, but enable predictions of which Sec14-like PtdIns/PtdCho transfer proteins are likely to be NPPM resistant or sensitive based on primary sequence considerations. Finally, the data provide independent evidence in support of previous studies highlighting the importance of Sec14 residue Ser173 in the mechanism by which NPPMs engage and inhibit Sec14-like PITPs.

  2. Regulation of primary metabolic pathways in oyster mushroom mycelia induced by blue light stimulation: accumulation of shikimic acid.

    PubMed

    Kojima, Masanobu; Kimura, Ninako; Miura, Ryuhei

    2015-02-27

    Shikimic acid is a key intermediate in the aromatic amino acid pathway as well as an important starting material for the synthesis of Tamiflu, a potent and selective inhibitor of the neuraminidase enzyme of influenza viruses A and B. Here we report that in oyster mushroom (Pleurotus ostreatus) mycelia cultivated in the dark, stimulation with blue light-emitting diodes induces the accumulation of shikimic acid. An integrated analysis of primary metabolites, gene expression and protein expression suggests that the accumulation of shikimic acid caused by blue light stimulation is due to an increase in 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (DAHPS, EC2.5.1.54), the rate-determining enzyme in the shikimic acid pathway, as well as phosphofructokinase (PFK, EC2.7.1.11) and glucose-6-phosphate dehydrogenase (G6PD, EC1.1.1.49), the rate-determining enzymes in the glycolysis and pentose phosphate pathways, respectively. This stimulation results in increased levels of phosphoenolpyruvic acid (PEP) and erythrose-4-phosphate (E4P), the starting materials of shikimic acid biosynthesis.

  3. Studies on the biochemistry and physiology of inositol phospholipids in Dunaliella salina

    SciTech Connect

    Einspahr, K.J.

    1988-01-01

    In the unicellular alga, Dunaliella salina, phosphatidylinositol, phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP{sub 2}) comprise 14.8, 1.2, and 0.3 mol %, respectively, of cellular phospholipids. In isolated plasma membrane fractions, PIP and PIP{sub 2} are highly concentrated, together comprising 9.5 mol % of plasmalemma phospholipids. The metabolism of these inositol phospholipids and phosphatidic acid (PA) is very rapid under normal growth conditions, as illustrated by the fact that within 5 minutes after introduction of {sup 32}P{sub i} into the growth medium over 75% of lipid-bound label was found in these quantitatively minor phospholipids. Within 2 minutes after a sudden hypoosmotic shock, the levels of PIP{sub 2} and PIP dropped to 65 and 79%, respectively, of controls. Within the same time frame PA rose to 141% of control values. These data suggest that a rapid breakdown of the polyphosphoinositides may mediate the profound morphological and physiological changes which allow this organism to survive drastic hypoosmotic stress.

  4. The acyltransferase LYCAT controls specific phosphoinositides and related membrane traffic

    PubMed Central

    Bone, Leslie N.; Dayam, Roya M.; Lee, Minhyoung; Kono, Nozomu; Fairn, Gregory D.; Arai, Hiroyuki; Botelho, Roberto J.; Antonescu, Costin N.

    2017-01-01

    Phosphoinositides (PIPs) are key regulators of membrane traffic and signaling. The interconversion of PIPs by lipid kinases and phosphatases regulates their functionality. Phosphatidylinositol (PI) and PIPs have a unique enrichment of 1-stearoyl-2-arachidonyl acyl species; however, the regulation and function of this specific acyl profile remains poorly understood. We examined the role of the PI acyltransferase LYCAT in control of PIPs and PIP-dependent membrane traffic. LYCAT silencing selectively perturbed the levels and localization of phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] and phosphatidylinositol-3-phosphate and the membrane traffic dependent on these specific PIPs but was without effect on phosphatidylinositol-4-phosphate or biosynthetic membrane traffic. The acyl profile of PI(4,5)P2 was selectively altered in LYCAT-deficient cells, whereas LYCAT localized with phosphatidylinositol synthase. We propose that LYCAT remodels the acyl chains of PI, which is then channeled into PI(4,5)P2. Our observations suggest that the PIP acyl chain profile may exert broad control of cell physiology. PMID:28035047

  5. Activation of TRPV1 channels inhibits mechanosensitive Piezo channel activity by depleting membrane phosphoinositides.

    PubMed

    Borbiro, Istvan; Badheka, Doreen; Rohacs, Tibor

    2015-02-10

    Capsaicin is an activator of the heat-sensitive TRPV1 (transient receptor potential vanilloid 1) ion channels and has been used as a local analgesic. We found that activation of TRPV1 channels with capsaicin either in dorsal root ganglion neurons or in a heterologous expression system inhibited the mechanosensitive Piezo1 and Piezo2 channels by depleting phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and its precursor phosphatidylinositol 4-phosphate [PI(4)P] from the plasma membrane through Ca(2+)-induced phospholipase Cδ (PLCδ) activation. Experiments with chemically inducible phosphoinositide phosphatases and receptor-induced activation of PLCβ indicated that inhibition of Piezo channels required depletion of both PI(4)P and PI(4,5)P2. The mechanically activated current amplitudes decreased substantially in the excised inside-out configuration, where the membrane patch containing Piezo1 channels is removed from the cell. PI(4,5)P2 and PI(4)P applied to these excised patches inhibited this decrease. Thus, we concluded that Piezo channel activity requires the presence of phosphoinositides, and the combined depletion of PI(4,5)P2 and PI(4)P reduces channel activity. In addition to revealing a role for distinct membrane lipids in mechanosensitive ion channel regulation, these data suggest that inhibition of Piezo2 channels may contribute to the analgesic effect of capsaicin.

  6. Dual roles for the Drosophila PI 4-kinase four wheel drive in localizing Rab11 during cytokinesis.

    PubMed

    Polevoy, Gordon; Wei, Ho-Chun; Wong, Raymond; Szentpetery, Zsofia; Kim, Yeun Ju; Goldbach, Philip; Steinbach, Sarah K; Balla, Tamas; Brill, Julie A

    2009-12-14

    Successful completion of cytokinesis relies on addition of new membrane, and requires the recycling endosome regulator Rab11, which localizes to the midzone. Despite the critical role of Rab11 in this process, little is known about the formation and composition of Rab11-containing organelles. Here, we identify the phosphatidylinositol (PI) 4-kinase III beta four wheel drive (Fwd) as a key regulator of Rab11 during cytokinesis in Drosophila melanogaster spermatocytes. We show Fwd is required for synthesis of PI 4-phosphate (PI4P) on Golgi membranes and for formation of PI4P-containing secretory organelles that localize to the midzone. Fwd binds and colocalizes with Rab11 on Golgi membranes, and is required for localization of Rab11 in dividing cells. A kinase-dead version of Fwd also binds Rab11 and partially restores cytokinesis to fwd mutant flies. Moreover, activated Rab11 partially suppresses loss of fwd. Our data suggest Fwd plays catalytic and noncatalytic roles in regulating Rab11 during cytokinesis.

  7. Phenyllactic acid production by simultaneous saccharification and fermentation of pretreated sorghum bagasse.

    PubMed

    Kawaguchi, Hideo; Teramura, Hiroshi; Uematsu, Kouji; Hara, Kiyotaka Y; Hasunuma, Tomohisa; Hirano, Ko; Sazuka, Takashi; Kitano, Hidemi; Tsuge, Yota; Kahar, Prihardi; Niimi-Nakamura, Satoko; Oinuma, Ken-ichi; Takaya, Naoki; Kasuga, Shigemitsu; Ogino, Chiaki; Kondo, Akihiko

    2015-04-01

    Dilute acid-pretreated sorghum bagasse, which was predominantly composed of glucan (59%) and xylose (7.2%), was used as a lignocellulosic feedstock for d-phenyllactic acid (PhLA) production by a recombinant Escherichia coli strain expressing phenylpyruvate reductase from Wickerhamia fluorescens. During fermentation with enzymatic hydrolysate of sorghum bagasse as a carbon source, the PhLA yield was reduced by 35% compared to filter paper hydrolysate, and metabolomics analysis revealed that NAD(P)H regeneration and intracellular levels of erythrose-4-phosphate and phosphoenolpyruvate for PhLA biosynthesis markedly reduced. Compared to separate hydrolysis and fermentation (SHF) with sorghum bagasse hydrolysate, simultaneous saccharification and fermentation (SSF) of sorghum bagasse under glucose limitation conditions yielded 4.8-fold more PhLA with less accumulation of eluted components, including p-coumaric acid and aldehydes, which inhibited PhLA fermentation. These results suggest that gradual enzymatic hydrolysis during SSF enhances PhLA production under glucose limitation and reduces the accumulation of fermentation inhibitors, collectively leading to increased PhLA yield.

  8. Cationic antimicrobial proteins isolated from human neutrophil granulocytes in the presence of diisopropyl fluorophosphate.

    PubMed Central

    Shafer, W M; Martin, L E; Spitznagel, J K

    1984-01-01

    Acid (0.2 M sodium acetate, pH 4.0) extracts of granules recovered from disrupted human polymorphonuclear granulocytes (PMNs) exhibited in vitro antimicrobial activity against Salmonella typhimurium. To minimize proteolytic destruction or modification of antimicrobial proteins derived from these granules, we pretreated the PMNs with the serine protease inhibitor diisopropyl fluorophosphate. Fractionation of such extracts by carboxymethyl Sephadex and Sephadex G-75 chromatography resulted in the recovery of at least two antimicrobial, cationic proteins. These proteins differed substantially in antimicrobial activity, amino acid composition, and molecular weight (Mr, 37,000 and 57,000). As we have shown before (Shafer et al., Infect. Immun. 43:834-858), with unfractionated proteins, these two proteins exhibited diminished activity against a polymyxin B-resistant (PBr) mutant of S. typhimurium compared with their activity against the isogenic parental polymyxin B-sensitive (PBs) strain. Expression of the relevant mutation (prmA) in the PBr mutant decreases the electronegativity of lipid A, owing to increased 4-amino-4-deoxy-L-arabinosylation at the 4' phosphate residue (Vaara et al., FEBS Lett. 129:145-149). The data suggest that at least two different cationic proteins account for the antimicrobial capacity of extracts from human PMN granules. Moreover, the availability of anionic charges in the outer membrane of S. typhimurium due to free lipid A phosphates apparently dictates phenotypic levels of resistance to both of the cationic proteins extracted from human PMN granules. Images PMID:6376359

  9. Lipid A and resistance of Salmonella typhimurium to antimicrobial granule proteins of human neutrophil granulocytes.

    PubMed Central

    Shafer, W M; Casey, S G; Spitznagel, J K

    1984-01-01

    Granule extracts from human polymorphonuclear leukocytes were prepared and fractionated by chromatography on Sephadex G75-SF. One fraction exhibited potent antimicrobial activity against an Rd1 lipopolysaccharide (LPS) mutant of Salmonella typhimurium. Susceptibility of the mutant to antimicrobial activity appeared to be due to binding of granule proteins to lipid A because isolated native LPS succeeded in blocking the antimicrobial activity of granule extracts whereas base-hydrolyzed LPS failed to do so. Centrifugation of control and base-hydrolyzed LPS-protein mixtures in cesium chloride gradients suggested that only control LPS formed complexes with antimicrobial proteins. Further evidence that bactericidal proteins from polymorphonuclear leukocyte granules interact with lipid A was that sublethal concentrations of polymyxin B (an antibiotic known to bind to lipid A) rendered target bacteria phenotypically resistant to granule proteins. Moreover, a mutant of S. typhimurium which synthesized a lipid A with decreased electronegativity due to increased 4-amino-4-deoxy-L-arabinosylation at the 4'-phosphate exhibited increased resistance to both polymyxin B and granule proteins. These results suggest that polymyxin B and antimicrobial proteins derived from polymorphonuclear leukocyte granules interact with lipid A in an analogous manner. PMID:6199303

  10. A metabolic node in action: chorismate-utilizing enzymes in microorganisms.

    PubMed

    Dosselaere, F; Vanderleyden, J

    2001-01-01

    The shikimate pathway has been described as a metabolic tree with many branches that led to the synthesis of an extensive range of products. This pathway is present only in bacteria, fungi, and plants. While there is only little difference in the sequence of the chemical reactions of the pathway, significant differences exist in terms of organization and regulation. In the main trunk of the shikimate pathway, D-erythrose 4-phosphate and phosphoenolpyruvate are converted via shikimate to chorismate. Chorismate is the common precursor for the biosynthesis of the aromatic amino acids, phenylalanine, tyrosine, and tryptophan, but also for other products as diverse as folate cofactors, benzoid and naphthoid coenzymes, phenazines, and siderophores. Five chorismate-utilizing enzymes have been characterized in microorganisms: chorismate mutase, anthranilate synthase, aminodeoxychorismate synthase, isochorismate synthase, and chorismate pyruvate-lyase. In this review these enzymes are discussed in terms of the corresponding gene structures and regulation, nucleotide and protein sequences, protein structures, and reaction mechanisms. The main emphasis is on transcriptional and posttranslational regulatory mechanisms, in view of how a microbial cell exploits its chorismate pool in diverse anabolic pathways. Comparison of the chorismate-utilizing enzymes has shown that some of them share sequence similarity, suggesting divergent evolution and commonality in reaction mechanisms. However, other chorismate-utilizing enzymes are examples of convergent evolution toward similar reaction capabilities.

  11. Mechanistic and inhibition studies of chorismate-utilizing enzymes.

    PubMed

    Kerbarh, O; Bulloch, E M M; Payne, R J; Sahr, T; Rébeillé, F; Abell, C

    2005-08-01

    The shikimate biosynthetic pathway is utilized in algae, higher plants, bacteria, fungi and apicomplexan parasites; it involves seven enzymatic steps in which phosphoenolpyruvate and erythrose 4-phosphate are converted into chorismate. In Escherichia coli, five chorismate-utilizing enzymes catalyse the synthesis of aromatic compounds such as L-phenylalanine, L-tyrosine, L-tryptophan, folate, ubiquinone and siderophores such as yersiniabactin and enterobactin. As mammals do not possess such a biosynthetic system, the enzymes involved in the pathway have aroused considerable interest as potential targets for the development of antimicrobial drugs and herbicides. As an initiative to investigate the mechanism of some of these enzymes, we showed that the antimicrobial effect of (6S)-6-fluoroshikimate is the result of irreversible inhibition of 4-amino-4-deoxychorismate synthase by 2-fluorochorismate. Based on this study, a catalytic mechanism for this enzyme was proposed, in which the residue Lys-274 is involved in the formation of a covalent intermediate. In another study, Yersinia enterocolitica Irp9, which is involved in the biosynthesis of the siderophore yersiniabactin, was for the first time biochemically characterized and shown to catalyse the formation of salicylate from chorismate via isochorismate as a reaction intermediate. A three-dimensional model for this enzyme was constructed that will guide the search for potent inhibitors of salicylate formation, and hence of bacterial iron uptake.

  12. OSBP-Related Protein Family in Lipid Transport Over Membrane Contact Sites

    PubMed Central

    Olkkonen, Vesa M.

    2015-01-01

    Increasing evidence suggests that oxysterol-binding protein-related proteins (ORPs) localize at membrane contact sites, which are high-capacity platforms for inter-organelle exchange of small molecules and information. ORPs can simultaneously associate with the two apposed membranes and transfer lipids across the interbilayer gap. Oxysterol-binding protein moves cholesterol from the endoplasmic reticulum to trans-Golgi, driven by the retrograde transport of phosphatidylinositol-4-phosphate (PI4P). Analogously, yeast Osh6p mediates the transport of phosphatidylserine from the endoplasmic reticulum to the plasma membrane in exchange for PI4P, and ORP5 and -8 are suggested to execute similar functions in mammalian cells. ORPs may share the capacity to bind PI4P within their ligand-binding domain, prompting the hypothesis that bidirectional transport of a phosphoinositide and another lipid may be a common theme among the protein family. This model, however, needs more experimental support and does not exclude a function of ORPs in lipid signaling. PMID:26715851

  13. [Properties of 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate--an intermediate in the non-mevalonate isoprenoid biosynthesis].

    PubMed

    Ostrovskiĭ, D N; Demina, G P; Deriabina, Iu I; Goncharenko, A V; Eberl, M; Shumaev, K B; Shashkov, A S

    2003-01-01

    Extraction and purification from the biomass of Corynebacterium ammoniagenes of 2-C-methyl-D-erhythritol 2,4-cyclopyrophosphate (MEC) was associated with its spontaneous transformation into a number of derivatives (which was due to pyrophosphate bond lability and the formation of complexes with metals). These derivatives included 1,2-cyclophospho-4-phosphate, 2,4-diphosphate, 2,3-cyclophosphate, 1,4-diphosphate, and 3,5-diphosphate (identified by 1H, 31P, and 13C NMR spectroscopy) and accounted for about 10% MEC. When added to a solution of DNA in the presence of the Fenton reagent, MEC prevented DNA decomposition. In addition, MEC slowed down the interaction of the reagent with tempol radicals, which indicates that complexation of ferrous ions by MEC attenuates their ability to catalyze the formation of hydroxyl radicals from hydrogen peroxide. In the presence of 0.23 mM MEC, the rate of respiration of rat liver mitochondria increased 1.8 times. At 0.1-1.0 mM, MEC activated in vitro proliferation of human Vgamma9 T-cells. It is suggested that MEC acts as an endogenous stabilizing agent for bacterial cells subjected to oxidative stress and as an immunomodulator for eukaryotic hosts.

  14. Influence of buffer composition on the distribution of inkjet printed protein molecules and the resulting spot morphology.

    PubMed

    Mujawar, Liyakat Hamid; van Amerongen, Aart; Norde, Willem

    2012-08-30

    Producing high quality protein microarrays on inexpensive substrates like polystyrene is a big challenge in the field of diagnostics. Using a non-contact inkjet printer we have produced microarrays on polystyrene slides for two different biotinylated biomolecules, bovine serum albumin (BSA-biotin) and immunoglobulin-G (IgG-biotin), and studied the influence of buffer (composition and pH) on the spot morphology and signal intensity. Atomic force microscopy revealed the morphological pattern of the (biomolecule) spots printed from phosphate buffer (pH 7.4), phosphate buffered saline (pH 7.4) and carbonate buffer (pH 9.6). The spots showed an irregular crust-like appearance when printed in phosphate buffered saline (pH 7.4), mainly due to the high NaCl content, whereas spots of biomolecules printed in carbonate buffer (pH 9.6) showed a smooth morphology. In addition, the rinsing of these dried spots led to the loss of a considerable fraction of the biomolecules, leaving behind a small fraction that is compatible with the (mono)layer. It was confirmed by confocal laser microscopy that the quality of the spots with respect to the uniformity and distribution of the biomolecules therein was superior when printed in carbonate buffer (pH 9.6) as compared to other buffer systems. Particularly, spotting in PBS yielded spots having a very irregular distribution and morphology.

  15. Resveratrol inhibits polyphosphoinositide metabolism in activated platelets.

    PubMed

    Olas, Beata; Wachowicz, Barbara; Holmsen, Holm; Fukami, Miriam H

    2005-08-15

    The effects of resveratrol (trans-3,4',5-trihydroxystilbene) on activation responses and the polyphosphoinositide metabolism in human blood platelets have been studied. Resveratrol partially inhibited secretory responses (liberation of dense granule nucleotides and lysosomal acid hydrolases), microparticle formation and protein phosphorylations induced by thrombin. The effects of resveratrol on phosphoinositide metabolites, phosphatidate (PtdOH), phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate (PtdIns-4(5)-P), phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2), phosphatidylinositol-3,4-bisphosphate (PtdIns-3,4-P2) and phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3) were monitored in blood platelets prelabelled with [32P]Pi. Resveratrol not only inhibited the marked increase in levels of PtdOH in platelets activated by thrombin (0.1 U/ml) but it decreased the steady state levels of the other polyphosphoinositide metabolites. The distribution of 32P in phosphoinositides in activated platelets was consistent with inhibition of CDP-DAG inositol transferase and a weak inhibition of PtdIns-4(5)-P kinase. These observations show that resveratrol has a profound effect on phospholipids, particularly on polyphosphoinositide metabolism, and may decrease the amount of PtdIns-4,5-P2 available for signalling in these cells.

  16. Mechanism of substrate specificity of phosphatidylinositol phosphate kinases

    PubMed Central

    Muftuoglu, Yagmur; Xue, Yi; Gao, Xiang; Wu, Dianqing; Ha, Ya

    2016-01-01

    The phosphatidylinositol phosphate kinase (PIPK) family of enzymes is primarily responsible for converting singly phosphorylated phosphatidylinositol derivatives to phosphatidylinositol bisphosphates. As such, these kinases are central to many signaling and membrane trafficking processes in the eukaryotic cell. The three types of phosphatidylinositol phosphate kinases are homologous in sequence but differ in catalytic activities and biological functions. Type I and type II kinases generate phosphatidylinositol 4,5-bisphosphate from phosphatidylinositol 4-phosphate and phosphatidylinositol 5-phosphate, respectively, whereas the type III kinase produces phosphatidylinositol 3,5-bisphosphate from phosphatidylinositol 3-phosphate. Based on crystallographic analysis of the zebrafish type I kinase PIP5Kα, we identified a structural motif unique to the kinase family that serves to recognize the monophosphate on the substrate. Our data indicate that the complex pattern of substrate recognition and phosphorylation results from the interplay between the monophosphate binding site and the specificity loop: the specificity loop functions to recognize different orientations of the inositol ring, whereas residues flanking the phosphate binding Arg244 determine whether phosphatidylinositol 3-phosphate is exclusively bound and phosphorylated at the 5-position. This work provides a thorough picture of how PIPKs achieve their exquisite substrate specificity. PMID:27439870

  17. Dynamic formation of ER–PM junctions presents a lipid phosphatase to regulate phosphoinositides

    PubMed Central

    Jensen, Jill B.; Vivas, Oscar; Kruse, Martin; Traynor-Kaplan, Alexis E.; Hille, Bertil

    2016-01-01

    Endoplasmic reticulum–plasma membrane (ER–PM) contact sites play an integral role in cellular processes such as excitation–contraction coupling and store-operated calcium entry (SOCE). Another ER–PM assembly is one tethered by the extended synaptotagmins (E-Syt). We have discovered that at steady state, E-Syt2 positions the ER and Sac1, an integral ER membrane lipid phosphatase, in discrete ER–PM junctions. Here, Sac1 participates in phosphoinositide homeostasis by limiting PM phosphatidylinositol 4-phosphate (PI(4)P), the precursor of PI(4,5)P2. Activation of G protein–coupled receptors that deplete PM PI(4,5)P2 disrupts E-Syt2–mediated ER–PM junctions, reducing Sac1’s access to the PM and permitting PM PI(4)P and PI(4,5)P2 to recover. Conversely, depletion of ER luminal calcium and subsequent activation of SOCE increases the amount of Sac1 in contact with the PM, depleting PM PI(4)P. Thus, the dynamic presence of Sac1 at ER–PM contact sites allows it to act as a cellular sensor and controller of PM phosphoinositides, thereby influencing many PM processes. PMID:27044890

  18. Fossilized microorganisms from the Emperor Seamounts: implications for the search for a subsurface fossil record on Earth and Mars.

    PubMed

    Ivarsson, M; Lausmaa, J; Lindblom, S; Broman, C; Holm, N G

    2008-12-01

    We have observed filamentous carbon-rich structures in samples drilled at 3 different seamounts that belong to the Emperor Seamounts in the Pacific Ocean: Detroit (81 Ma), Nintoku (56 Ma), and Koko Seamounts (48 Ma). The samples consist of low-temperature altered basalts recovered from all 3 seamounts. The maximum depth from which the samples were retrieved was 954 meters below seafloor (mbsf). The filamentous structures occur in veins and fractures in the basalts, where they are attached to the vein walls and embedded in vein-filling minerals like calcite, aragonite, and gypsum. The filaments were studied with a combination of optical microscopy, environmental scanning electron microscopy (ESEM), Raman spectroscopy, and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Minerals were identified by a combination of optical microscopy, X-ray diffraction, Raman spectrometry, and energy dispersive spectrometry on an environmental scanning electron microscope. Carbon content of the filaments ranges between approximately 10 wt % and approximately 50 wt % and is not associated with carbonates. These results indicate an organic origin of the carbon. The presence of C(2)H(4), phosphate, and lipid-like molecules in the filaments further supports a biogenic origin. We also found microchannels in volcanic glass enriched in carbon (approximately 10-40 wt %) compatible with putative microbial activity. Our findings suggest new niches for life in subseafloor environments and have implications for further exploration of the subseafloor biosphere on Earth and beyond.

  19. Enhancing Nanoparticle Accumulation and Retention in Desmoplastic Tumors via Vascular Disruption for Internal Radiation Therapy

    PubMed Central

    Satterlee, Andrew B.; Rojas, Juan D.; Dayton, Paul A.; Huang, Leaf

    2017-01-01

    Aggressive, desmoplastic tumors are notoriously difficult to treat because of their extensive stroma, high interstitial pressure, and resistant tumor microenvironment. We have developed a combination therapy that can significantly slow the growth of large, stroma-rich tumors by causing massive apoptosis in the tumor center while simultaneously increasing nanoparticle uptake through a treatment-induced increase in the accumulation and retention of nanoparticles in the tumor. The vascular disrupting agent Combretastatin A-4 Phosphate (CA4P) is able to increase the accumulation of radiation-containing nanoparticles for internal radiation therapy, and the retention of these delivered radioisotopes is maintained over several days. We use ultrasound to measure the effect of CA4P in live tumor-bearing mice, and we encapsulate the radio-theranostic isotope 177Lutetium as a therapeutic agent as well as a means to measure nanoparticle accumulation and retention in the tumor. This combination therapy induces prolonged apoptosis in the tumor, decreasing both the fibroblast and total cell density and allowing further tumor growth inhibition using a cisplatin-containing nanoparticle. PMID:28042332

  20. Collagen-induced binding to human platelets of platelet-derived growth factor leading to inhibition of P43 and P20 phosphorylation

    SciTech Connect

    Bryckaert, M.C.; Rendu, F.; Tobelem, G.; Wasteson, A.

    1989-03-15

    Platelet-derived growth factor (PDGF) is known to inhibit collagen-induced platelet aggregation. Collagen-induced binding of /sup 125/I-PDGF to human washed platelets was therefore investigated. It was found to be time-dependent, reaching a plateau at 20 degrees C after 30 min, collagen concentration-dependent, specifically inhibited by unlabeled PDGF, and saturable. Scatchard plot analysis showed a single class of sites with 3000 +/- 450 molecules bound/cell and an apparent KD of 1.2 +/- 0.2 10(-8) M. The effects of PDGF on collagen-induced phosphoinositide breakdown and protein phosphorylation were also investigated. At 50 ng/ml PDGF, a concentration which completely inhibited collagen-induced aggregation, the breakdown of (/sup 32/P)phosphatidylinositol 4,5-biphosphate (PIP2) and (/sup 32/P)phosphatidylinositol 4-phosphate (PIP) was observed, but the subsequent replenishment of (/sup 32/P)PIP2 was inhibited. The same PDGF concentration totally inhibited collagen-induced phosphatidic acid formation. PDGF also completely prevented phosphorylation of P43 and P20, as a result of protein kinase C activation consecutive to phosphoinositide metabolism. These results suggest that a specific PDGF receptor can be induced by collagen, and PDGF can effect the early events of collagen-induced platelet activation by inhibiting PIP2 resynthesis and P43 and P20 phosphorylation. It is concluded that PDGF might be involved in a negative feed-back control of platelet activation.

  1. Chronotherapeutic drug delivery of Tamarind gum, Chitosan and Okra gum controlled release colon targeted directly compressed Propranolol HCl matrix tablets and in-vitro evaluation.

    PubMed

    Newton, A M J; Indana, V L; Kumar, Jatinder

    2015-08-01

    The main objective of this investigation is to develop a chronotherapeutic drug delivery of various natural polymers based colon targeted drug delivery systems to treat early morning sign in BP. The polymers such as Tamarind gum, Okra gum and Chitosan were used in the formulation design. A model drug Propranolol HCl was incorporated in the formulation in order to assess the controlled release and time dependent release potential of various natural polymers. A novel polymer Tamarind gum was extracted and used as a prime polymer in this study to prove the superiority of this polymer over other leading natural polymer. Propranolol HCl was used as a model drug which undergoes hepatic metabolism and witnesses the poor bioavailability. The matrix tablets of Propranolol HCl were prepared by direct compression. The tablets were evaluated for various quality control parameters and found to be within the limits. Carbopol 940 was used as an auxiliary polymer to modify the drug release and physicochemical characteristics of the tablets. The in vitro release studies were performed in 0.1N HCl for 1.5h, followed by pH 6.8 phosphate buffer for 2h and pH 7.4 phosphate buffer till maximum amount of drug release. The in vitro release profile of the formulations were fitted with various pharmacokinetic mathematical models and analyzed for release profile. The formulations prepared with Tamarind gum prolonged the release for an extended period of time compared to other polymer based formulation and showed an excellent compression characteristic.

  2. The shikimate pathway: review of amino acid sequence, function and three-dimensional structures of the enzymes.

    PubMed

    Mir, Rafia; Jallu, Shais; Singh, T P

    2015-06-01

    The aromatic compounds such as aromatic amino acids, vitamin K and ubiquinone are important prerequisites for the metabolism of an organism. All organisms can synthesize these aromatic metabolites through shikimate pathway, except for mammals which are dependent on their diet for these compounds. The pathway converts phosphoenolpyruvate and erythrose 4-phosphate to chorismate through seven enzymatically catalyzed steps and chorismate serves as a precursor for the synthesis of variety of aromatic compounds. These enzymes have shown to play a vital role for the viability of microorganisms and thus are suggested to present attractive molecular targets for the design of novel antimicrobial drugs. This review focuses on the seven enzymes of the shikimate pathway, highlighting their primary sequences, functions and three-dimensional structures. The understanding of their active site amino acid maps, functions and three-dimensional structures will provide a framework on which the rational design of antimicrobial drugs would be based. Comparing the full length amino acid sequences and the X-ray crystal structures of these enzymes from bacteria, fungi and plant sources would contribute in designing a specific drug and/or in developing broad-spectrum compounds with efficacy against a variety of pathogens.

  3. Autophagic lysosomal reformation depends on mTOR reactivation in H2O2-induced autophagy.

    PubMed

    Zhang, Jiqian; Zhou, Wei; Lin, Jun; Wei, Pengfei; Zhang, Yunjiao; Jin, Peipei; Chen, Ming; Man, Na; Wen, Longping

    2016-01-01

    Autophagic lysosomal reformation, a key cellular process for maintaining lysosome homeostasis in elevated autophagy, so far has only been reported for cells under certain forms of starvation. For this reason, it is controversial that whether this phenomenon is starvation-specific and its importance in lysosomal regeneration at the late stage of autophagy is often challenged. Here we show that exogenous hydrogen peroxide (H2O2) induced lysosome depletion and recovery characteristic of autophagic lysosomal reformation, and we confirmed the occurrence of autophagic lysosomal reformation after H2O2 treatment by demonstrating Rab7 dissociation from autolysosomes, recruitment of Phosphatidylinositol 4-phosphate (PI4P) and clathrin to the surface of autolysosomes, and the existence of tubular "pro-lysosome" structures extending from autolysosomes. Similar to starvation, H2O2 caused an initial deactivation and a subsequent reactivation for mTOR, and mTOR reactivation was essential for ALR. Our results provided a first non-starvation example of autophagic lysosomal reformation and provide evidence for its importance for some autophagic processes other than that of starvation.

  4. De Novo Transcriptome and Expression Profile Analysis to Reveal Genes and Pathways Potentially Involved in Cantharidin Biosynthesis in the Blister Beetle Mylabris cichorii

    PubMed Central

    Huang, Yi; Wang, Zhongkang; Zha, Shenfang; Wang, Yu; Jiang, Wei; Liao, Yufeng; Song, Zhangyong; Qi, Zhaoran; Yin, Youping

    2016-01-01

    The dried body of Mylabris cichorii is well-known Chinese traditional medicine. The sesquiterpenoid cantharidin, which is secreted mostly by adult male beetles, has recently been used as an anti-cancer drug. However, little is known about the mechanisms of cantharidin biosynthesis. Furthermore, there is currently no genomic or transcriptomic information for M. cichorii. In this study, we performed de novo assembly transcriptome of M. cichorii using the Illumina Hiseq2000. A single run produced 9.19 Gb of clean nucleotides comprising 29,247 sequences, including 23,739 annotated sequences (about 81%). We also constructed two expression profile libraries (20–25 day-old adult males and 20–25 day-old adult females) and discovered 2,465 significantly differentially-expressed genes. Putative genes and pathways involved in the biosynthesis of cantharidin were then characterized. We also found that cantharidin biosynthesis in M. cichorii might only occur via the mevalonate (MVA) pathway, not via the methylerythritol 4-phosphate/deoxyxylulose 5-phosphate (MEP/DOXP) pathway or a mixture of these. Besides, we considered that cantharidin biosynthesis might be related to the juvenile hormone (JH) biosynthesis or degradation. The results of transcriptome and expression profiling analysis provide a comprehensive sequence resource for M. cichorii that could facilitate the in-depth study of candidate genes and pathways involved in cantharidin biosynthesis, and may thus help to improve our understanding of the mechanisms of cantharidin biosynthesis in blister beetles. PMID:26752526

  5. Two-ligand priming mechanism for potentiated phosphoinositide synthesis is an evolutionarily conserved feature of Sec14-like phosphatidylinositol and phosphatidylcholine exchange proteins

    PubMed Central

    Huang, Jin; Ghosh, Ratna; Tripathi, Ashutosh; Lönnfors, Max; Somerharju, Pentti; Bankaitis, Vytas A.

    2016-01-01

    Lipid signaling, particularly phosphoinositide signaling, plays a key role in regulating the extreme polarized membrane growth that drives root hair development in plants. The Arabidopsis AtSFH1 gene encodes a two-domain protein with an amino-terminal Sec14-like phosphatidylinositol transfer protein (PITP) domain linked to a carboxy-terminal nodulin domain. AtSfh1 is critical for promoting the spatially highly organized phosphatidylinositol-4,5-bisphosphate signaling program required for establishment and maintenance of polarized root hair growth. Here we demonstrate that, like the yeast Sec14, the AtSfh1 PITP domain requires both its phosphatidylinositol (PtdIns)- and phosphatidylcholine (PtdCho)-binding properties to stimulate PtdIns-4-phosphate [PtdIns(4)P] synthesis. Moreover, we show that both phospholipid-binding activities are essential for AtSfh1 activity in supporting polarized root hair growth. Finally, we report genetic and biochemical evidence that the two-ligand mechanism for potentiation of PtdIns 4-OH kinase activity is a broadly conserved feature of plant Sec14-nodulin proteins, and that this strategy appeared only late in plant evolution. Taken together, the data indicate that the PtdIns/PtdCho-exchange mechanism for stimulated PtdIns(4)P synthesis either arose independently during evolution in yeast and in higher plants, or a suitable genetic module was introduced to higher plants from a fungal source and subsequently exploited by them. PMID:27193303

  6. Sphingolipid transfer proteins defined by the GLTP-fold

    PubMed Central

    Malinina, Lucy; Simanshu, Dhirendra K.; Zhai, Xiuhong; Samygina, Valeria R.; Kamlekar, RaviKanth; Kenoth, Roopa; Ochoa-Lizarralde, Borja; Malakhova, Margarita L.; Molotkovsky, Julian G.; Patel, Dinshaw J.; Brown, Rhoderick E.

    2015-01-01

    Glycolipid transfer proteins (GLTPs) originally were identified as small (~24 kDa), soluble, amphitropic proteins that specifically accelerate the intermembrane transfer of glycolipids. GLTPs and related homologs now are known to adopt a unique, helically dominated, two-layer ‘sandwich’ architecture defined as the GLTP-fold that provides the structural underpinning for the eukaryotic GLTP superfamily. Recent advances now provide exquisite insights into structural features responsible for lipid headgroup selectivity as well as the adaptability of the hydrophobic compartment for accommodating hydrocarbon chains of differing length and unsaturation. A new understanding of the structural versatility and evolutionary premium placed on the GLTP motif has emerged. Human GLTP-motifs have evolved to function not only as glucosylceramide binding/transferring domains for phosphoinositol 4-phosphate adaptor protein-2 during glycosphingolipid biosynthesis but also as selective binding/transfer proteins for ceramide-1-phosphate. The latter, known as ceramide-l-phosphate transfer protein, recently has been shown to form GLTP-fold while critically regulating Group-IV cytoplasmic phospholipase A2 activity and pro-inflammatory eicosanoid production. PMID:25797198

  7. Carotenoid Derivates in Achiote (Bixa orellana) Seeds: Synthesis and Health Promoting Properties

    PubMed Central

    Rivera-Madrid, Renata; Aguilar-Espinosa, Margarita; Cárdenas-Conejo, Yair; Garza-Caligaris, Luz E.

    2016-01-01

    Bixa orellana (family Bixaceae) is a neotropical fast growing perennial tree of great agro-industrial value because its seeds have a high carotenoid content, mainly bixin. It has been used since pre-colonial times as a culinary colorant and spice, and for healing purposes. It is currently used as a natural pigment in the food, in pharmaceutical, and cosmetic industries, and it is commercially known as annatto. Recently, several studies have addressed the biological and medical properties of this natural pigment, both as potential source of new drugs or because its ingestion as a condiment or diet supplement may protect against several diseases. The most documented properties are anti-oxidative; but its anti-cancer, hypoglucemic, antibiotic and anti-inflammatory properties are also being studied. Bixin’s pathway elucidation and its regulation mechanisms are critical to improve the produce of this important carotenoid. Even though the bixin pathway has been established, the regulation of the genes involved in bixin production remains largely unknown. Our laboratory recently published B. orellana’s transcriptome and we have identified most of its MEP (methyl-D-erythritol 4-phosphate) and carotenoid pathway genes. Annatto is a potential source of new drugs and can be a valuable nutraceutical supplement. However, its nutritional and healing properties require further study. PMID:27708658

  8. Molecular cloning, functional characterization and expression of potato (Solanum tuberosum) 1-deoxy-d-xylulose 5-phosphate synthase 1 (StDXS1) in response to Phytophthora infestans.

    PubMed

    Henriquez, Maria Antonia; Soliman, Atta; Li, Genyi; Hannoufa, Abdelali; Ayele, Belay T; Daayf, Fouad

    2016-02-01

    1-Deoxy-D-xylulose 5-phosphate synthase (DXS) catalyzes the initial step of the plastidial 2C-methyl-D-erythritol-4-phosphate (DOXP-MEP) pathway involved in isoprenoid biosynthesis. In this study, we cloned the complete cDNA of potato DXS gene that was designated StDXS1. StDXS1 cDNA encodes for 719 amino acid residues, with MW of 77.8 kDa, and is present in one copy in the potato genome. Phylogenetic analysis and protein sequence alignments assigned StDXS1 to a group with DXS homologues from closely related species and exhibited homodomain identity with known DXS proteins from other plant species. Late blight symptoms occurred in parallel with a reduction in StDXS1 transcript levels, which may be associated with the levels of isoprenoids that contribute to plant protection against pathogens. Subcellular localization indicated that StDXS1 targets the chloroplasts where isoprenoids are synthesized. Arabidopsis expressing StDXS1 showed a higher accumulation of carotenoids and chlorophyll as compared to wild type controls. Lower levels of ABA and GA were detected in the transgenic DXS lines as compared to control plants, which reflected on higher germination rates of the transgenic DXS lines. No changes were detected in JA or SA contents. Selected downstream genes in the DOXP-MEP pathway, especially GGPPS genes, were up-regulated in the transgenic lines.

  9. Design, Synthesis, in Vitro, and in Vivo Anticancer and Antiangiogenic Activity of Novel 3-Arylaminobenzofuran Derivatives Targeting the Colchicine Site on Tubulin

    PubMed Central

    Romagnoli, Romeo; Baraldi, Pier Giovanni; Salvador, Maria Kimatrai; Prencipe, Filippo; Lopez-Cara, Carlota; Ortega, Santiago Schiaffino; Brancale, Andrea; Hamel, Ernest; Castagliuolo, Ignazio; Mitola, Stefania; Ronca, Roberto; Bortolozzi, Roberta; Porcù, Elena; Basso, Giuseppe; Viola, Giampietro

    2015-01-01

    A new series of compounds characterized by the presence of a 2-methoxy/ethoxycarbonyl group, combined with either no substituent or a methoxy group at each of the four possible positions of the benzene portion of the 3-(3′,4′,5′-trimethoxyanilino)benzo[b]furan skeleton, were evaluated for antiproliferative activity against cancer cells in culture and, for selected, highly active compounds, inhibition of tubulin polymerization, cell cycle effects, and in vivo potency. The greatest antiproliferative activity occurred with a methoxy group introduced at the C-6 position, the least with this substituent at C-4. Thus far, the most promising compound in this series was 2-methoxycarbonyl-3-(3′,4′,5′-trimethoxyanilino)-6-methoxybenzo-[b]furan (3g), which inhibited cancer cell growth at nanomolar concentrations (IC50 values of 0.3–27 nM), bound to the colchicine site of tubulin, induced apoptosis, and showed, both in vitro and in vivo, potent vascular disrupting properties derived from the effect of this compound on vascular endothelial cells. Compound 3g had in vivo antitumor activity in a murine model comparable to the activity obtained with combretastatin A-4 phosphate. PMID:25785605

  10. Brain polyphosphoinositide metabolism during focal ischemia in rat cortex

    SciTech Connect

    Lin, T.N.; Liu, T.H.; Xu, J.; Hsu, C.Y.; Sun, G.Y. )

    1991-04-01

    Using a rat model of stroke, we examined the effects of focal cerebral ischemia on the metabolism of polyphosphoinositides by injecting {sup 32}Pi into both the left and right cortices. After equilibration of the label for 2-3 hours, ischemia induced a significant decrease (p less than 0.001) in the concentrations of labeled phosphatidyl 4,5-bisphosphates (66-78%) and phosphatidylinositol 4-phosphate (64-67%) in the right middle cerebral artery cortex of four rats. The phospholipid labeling pattern in the left middle cerebral artery cortex, which sustained only mild ischemia and no permanent tissue damage, was not different from that of two sham-operated controls. However, when {sup 32}Pi was injected 1 hour after the ischemic insult, there was a significant decrease (p less than 0.01) in the incorporation of label into the phospholipids in both cortices of four ischemic rats compared with four sham-operated controls. Furthermore, differences in the phospholipid labeling pattern were observed in the left cortex compared with the sham-operated controls. The change in labeling pattern was attributed to the partial reduction in blood flow following ligation of the common carotid arteries. We provide a sensitive procedure for probing the effects of focal cerebral ischemia on the polyphosphoinositide signaling pathway in the brain, which may play an important role in the pathogenesis of tissue injury.

  11. Regulation of the actin cytoskeleton by PIP2 in cytokinesis.

    PubMed

    Logan, Michael R; Mandato, Craig A

    2006-06-01

    Cytokinesis is a sequential process that occurs in three phases: assembly of the cytokinetic apparatus, furrow progression and fission (abscission) of the newly formed daughter cells. The ingression of the cleavage furrow is dependent on the constriction of an equatorial actomyosin ring in many cell types. Recent studies have demonstrated that this structure is highly dynamic and undergoes active polymerization and depolymerization throughout the furrowing process. Despite much progress in the identification of contractile ring components, little is known regarding the mechanism of its assembly and structural rearrangements. PIP2 (phosphatidylinositol 4,5-bisphosphate) is a critical regulator of actin dynamics and plays an essential role in cell motility and adhesion. Recent studies have indicated that an elevation of PIP2 at the cleavage furrow is a critical event for furrow stability. In this review we discuss the role of PIP2-mediated signalling in the structural maintenance of the contractile ring and furrow progression. In addition, we address the role of other phosphoinositides, PI(4)P (phosphatidylinositol 4-phosphate) and PIP3 (phosphatidylinositol 3,4,5-triphosphate) in these processes.

  12. Rab1 interacts with GOLPH3 and controls Golgi structure and contractile ring constriction during cytokinesis in Drosophila melanogaster

    PubMed Central

    Sechi, Stefano; Frappaolo, Anna; Fraschini, Roberta; Capalbo, Luisa; Gottardo, Marco; Belloni, Giorgio; Glover, David M.

    2017-01-01

    Cytokinesis requires a tight coordination between actomyosin ring constriction and new membrane addition along the ingressing cleavage furrow. However, the molecular mechanisms underlying vesicle trafficking to the equatorial site and how this process is coupled with the dynamics of the contractile apparatus are poorly defined. Here we provide evidence for the requirement of Rab1 during cleavage furrow ingression in cytokinesis. We demonstrate that the gene omelette (omt) encodes the Drosophila orthologue of human Rab1 and is required for successful cytokinesis in both mitotic and meiotic dividing cells of Drosophila melanogaster. We show that Rab1 protein colocalizes with the conserved oligomeric Golgi (COG) complex Cog7 subunit and the phosphatidylinositol 4-phosphate effector GOLPH3 at the Golgi stacks. Analysis by transmission electron microscopy and 3D-SIM super-resolution microscopy reveals loss of normal Golgi architecture in omt mutant spermatocytes indicating a role for Rab1 in Golgi formation. In dividing cells, Rab1 enables stabilization and contraction of actomyosin rings. We further demonstrate that GTP-bound Rab1 directly interacts with GOLPH3 and controls its localization at the Golgi and at the cleavage site. We propose that Rab1, by associating with GOLPH3, controls membrane trafficking and contractile ring constriction during cytokinesis. PMID:28100664

  13. Metabolic Engineering of Pseudomonas putida KT2440 for the Production of para-Hydroxy Benzoic Acid

    PubMed Central

    Yu, Shiqin; Plan, Manuel R.; Winter, Gal; Krömer, Jens O.

    2016-01-01

    para-Hydroxy benzoic acid (PHBA) is the key component for preparing parabens, a common preservatives in food, drugs, and personal care products, as well as high-performance bioplastics such as liquid crystal polymers. Pseudomonas putida KT2440 was engineered to produce PHBA from glucose via the shikimate pathway intermediate chorismate. To obtain the PHBA production strain, chorismate lyase UbiC from Escherichia coli and a feedback resistant 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase encoded by gene aroGD146N were overexpressed individually and simultaneously. In addition, genes related to product degradation (pobA) or competing for the precursor chorismate (pheA and trpE) were deleted from the genome. To further improve PHBA production, the glucose metabolism repressor hexR was knocked out in order to increase erythrose 4-phosphate and NADPH supply. The best strain achieved a maximum titer of 1.73 g L−1 and a carbon yield of 18.1% (C-mol C-mol−1) in a non-optimized fed-batch fermentation. This is to date the highest PHBA concentration produced by P. putida using a chorismate lyase. PMID:27965953

  14. Effect of hydration on the permeation of ketoconazole through human nail plate in vitro.

    PubMed

    Gunt, Hemali B; Kasting, Gerald B

    2007-12-01

    The impact of hydration on the permeation of the antifungal drug, ketoconazole, through excised human nails in vitro was evaluated in diffusion cell studies. Nails treated with [(3)H]ketoconazole solvent-deposited onto the dorsal surface were maintained in incubators at 32 degrees C and exposed sequentially to relative humidities (dorsal side) of 15, 40, 80 and 100% over a period of 40 days. The ventral side was bathed in a pH 7.4 phosphate buffer. Ascending and descending humidity regimens were tested. Increasing the ambient RH from 15 to 100% enhanced permeation of radiolabel associated with [(3)H]ketoconazole by a factor of three. Diffusivities estimated from these data and the associated nail water contents (estimated in a separate study) can be described by a free volume theory. Therefore, formulations or treatments, which increase nail hydration, have potential to improve topical therapy for onychomycosis, if a favorable balance between drug delivery and growth conditions for the dermatophytes can be achieved.

  15. Arenavirus Infection Induces Discrete Cytosolic Structures for RNA Replication

    PubMed Central

    Baird, Nicholas L.; York, Joanne

    2012-01-01

    Arenaviruses are responsible for acute hemorrhagic fevers with high mortality and pose significant threats to public health and biodefense. These enveloped negative-sense RNA viruses replicate in the cell cytoplasm and express four proteins. To better understand how these proteins insinuate themselves into cellular processes to orchestrate productive viral replication, we have identified and characterized novel cytosolic structures involved in arenavirus replication and transcription. In cells infected with the nonpathogenic Tacaribe virus or the attenuated Candid#1 strain of Junín virus, we find that newly synthesized viral RNAs localize to cytosolic puncta containing the nucleoprotein (N) of the virus. Density gradient centrifugation studies reveal that these replication-transcription complexes (RTCs) are associated with cellular membranes and contain full-length genomic- and antigenomic-sense RNAs. Viral mRNAs segregate at a higher buoyant density and are likewise scant in immunopurified RTCs, consistent with their translation on bulk cellular ribosomes. In addition, confocal microscopy analysis reveals that RTCs contain the lipid phosphatidylinositol-4-phosphate and proteins involved in cellular mRNA metabolism, including the large and small ribosomal subunit proteins L10a and S6, the stress granule protein G3BP1, and a subset of translation initiation factors. Elucidating the structure and function of RTCs will enhance our understanding of virus-cell interactions that promote arenavirus replication and mitigate against host cell immunity. This knowledge may lead to novel intervention strategies to limit viral virulence and pathogenesis. PMID:22875974

  16. Synthetic growth phenotypes of E.coli lacking ppGpp and transketolase A (tktA) are due to ppGpp-mediated transcriptional regulation of tktB

    PubMed Central

    Harinarayanan, Rajendran; Murphy, Helen; Cashel, Michael

    2008-01-01

    Summary Many physiological adjustments to nutrient changes involve ppGpp. Recent attempts to deduce ppGpp regulatory effects using proteomics or gene profiling can rigorously identify proteins or transcripts, but the functional significance is often unclear. Using a random screen for synthetic lethals we found a ppGpp-dependent functional pathway that operates through transketolase-B (TktB), and which is “buffered” in wildtype strain by the presence of an isozyme, transketolase-A (TktA). Transketolase activity is required in cells to make erythrose-4-phosphate, a precursor of aromatic amino acids and vitamins. By studying tktB-dependent nutritional requirements as well as measuring activities using PtalA-tktB′-lacZ transcriptional reporter fusion we show positive transcriptional regulation of the talA-tktB operon by ppGpp. Our results show the existence of RpoS-dependent and independent modes of positive regulation by ppGpp. Both routes of activation are magnified by elevating ppGpp levels with a spoT mutation (spoT-R39A) defective in hydrolase but not synthetase activity or with the stringent suppressor mutations rpoB-A532Δ or rpoB-T563P in the absence of ppGpp. PMID:18532980

  17. Effects of fosmidomycin on plant photosynthesis as measured by gas exchange and chlorophyll fluorescence.

    PubMed

    Possell, Malcolm; Ryan, Annette; Vickers, Claudia E; Mullineaux, Philip M; Hewitt, C Nicholas

    2010-04-01

    In higher plants, many isoprenoids are synthesised via the chloroplastic 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Attempts to elucidate the function of individual isoprenoids have used the antibiotic/herbicidal compound fosmidomycin (3-[N-formyl-N-hydroxy amino] propyl phosphonic acid) to inhibit this pathway. Examination of the effect of fosmidomycin on the major components of photosynthesis in leaves of white poplar (Populus alba) and tobacco (Nicotiana tabacum) was made. Fosmidomycin reduced net photosynthesis in both species within 1 h of application, but only when photosynthesis was light-saturated. In P. alba, these reductions were confounded by high light and fosmidomycin inducing stomatal patchiness. In tobacco, this was caused by significant reductions in PSII chlorophyll fluorescence and reductions in V(cmax) and J(max). Our data indicate that the diminution of photosynthesis is likely a complex effect resulting from the inhibition of multiple MEP pathway products, resulting in photoinhibition and photo-damage. These effects should be accounted for in experimental design and analysis when using fosmidomycin to avoid misinterpretation of results as measured by gas exchange and chlorophyll fluorescence.

  18. Identifying novel genes and chemicals related to nasopharyngeal cancer in a heterogeneous network.

    PubMed

    Li, Zhandong; An, Lifeng; Li, Hao; Wang, ShaoPeng; Zhou, You; Yuan, Fei; Li, Lin

    2016-05-05

    Nasopharyngeal cancer or nasopharyngeal carcinoma (NPC) is the most common cancer originating in the nasopharynx. The factors that induce nasopharyngeal cancer are still not clear. Additional information about the chemicals or genes related to nasopharyngeal cancer will promote a better understanding of the pathogenesis of this cancer and the factors that induce it. Thus, a computational method NPC-RGCP was proposed in this study to identify the possible relevant chemicals and genes based on the presently known chemicals and genes related to nasopharyngeal cancer. To extensively utilize the functional associations between proteins and chemicals, a heterogeneous network was constructed based on interactions of proteins and chemicals. The NPC-RGCP included two stages: the searching stage and the screening stage. The former stage is for finding new possible genes and chemicals in the heterogeneous network, while the latter stage is for screening and removing false discoveries and selecting the core genes and chemicals. As a result, five putative genes, CXCR3, IRF1, CDK1, GSTP1, and CDH2, and seven putative chemicals, iron, propionic acid, dimethyl sulfoxide, isopropanol, erythrose 4-phosphate, β-D-Fructose 6-phosphate, and flavin adenine dinucleotide, were identified by NPC-RGCP. Extensive analyses provided confirmation that the putative genes and chemicals have significant associations with nasopharyngeal cancer.

  19. Structural Basis for PI(4)P-Specific Membrane Recruitment of the Legionella pneumophila Effector DrrA/SidM

    PubMed Central

    Del Campo, Claudia M.; Mishra, Ashwini K.; Wang, Yu-Hsiu; Roy, Craig R.; Janmey, Paul A.; Lambright, David G.

    2014-01-01

    SUMMARY Recruitment of the Legionella pneumophila effector DrrA to the Legionella-containing vacuole, where it activates and AMPylates Rab1, is mediated by a P4M domain that binds phosphatidylinositol 4-phosphate [PI(4)P] with high affinity and specificity. Despite the importance of PI(4)P in Golgi trafficking and its manipulation by pathogens, the structural bases for PI(4)P-dependent membrane recruitment remain poorly defined. Here, we determined the crystal structure of a DrrA fragment including the P4M domain in complex with dibutyl PI(4)P and investigated the determinants of phosphoinositide recognition and membrane targeting. Headgroup recognition involves an elaborate network of direct and water-mediated interactions with basic and polar residues in the context of a deep, constrictive binding pocket. An adjacent hydrophobic helical element packs against the acyl chains and inserts robustly into PI(4)P-containing monolayers. The structural, biochemical, and biophysical data reported here support a detailed structural mechanism for PI(4)P-dependent membrane targeting by DrrA. PMID:24530282

  20. Diverse communities of active Bacteria and Archaea along oxygen gradients in coral reef sediments

    NASA Astrophysics Data System (ADS)

    Rusch, A.; Hannides, A. K.; Gaidos, E.

    2009-03-01

    Microbial communities inhabiting highly permeable sediments of Checker Reef in Kaneohe Bay, Hawaii, were characterized in relation to porewater geochemistry (O2, NO3 -, NO2 -, NH4 +, phosphate). The physiologically active part of the population, assessed by sequencing cDNA libraries of 16S rRNA amplicons, was very diverse, with an estimated ribotype richness ≥1,380 in anoxic sediment. Quantitative analysis of community structure by rRNA-targeted fluorescence in situ hybridization (FISH) indicated that the archaeal population (9-18%) was dominated by marine Crenarchaeota (5-9%). Planctomycetales were the most abundant group in the oxic and interfacial habitat (17-19%) but were a minority (<5%) in anoxic reef sediment, where γ-Proteobacteria were numerically dominant (18%). Another 9-14% of the microbial benthos belonged to β-Proteobacteria, predominantly within the order Nitrosomonadales, many cultured representatives of which are NH4 + oxidizers. The results of this study contribute to the phylogenetic characterization of benthic microbial communities that are important in organic matter degradation and nutrient recycling in coral reef ecosystems.

  1. Annexin VI is attached to transverse-tubule membranes in isolated skeletal muscle triads.

    PubMed

    Barrientos, G; Hidalgo, C

    2002-07-15

    Annexin VI is a 68-kDa protein of the Annexin family, a group of Ca2+-dependent phospholipid-binding proteins widely distributed in mammalian tissues including skeletal muscle. We investigated a) which membrane system contributes Annexin VI to skeletal muscle triads, and b) whether Annexin VI removal affects triad integrity or function. Annexin VI was present in isolated triads and transverse tubules but not in heavy sarcoplasmic reticulum vesicles, indicating that Annexin VI binds to either free or triad-attached transverse tubules. Extraction with EGTA of Annexin VI from triads did not alter their migration as a single band in sucrose density gradients or their ouabain binding-site density, indicating that triad integrity does not require Annexin VI. Caffeine-induced Ca2+ release kinetics and Ca2+ uptake rates were likewise not affected by Annexin VI removal from triads, suggesting that Annexin VI is not involved in these functions. Annexin VI purified from rabbit skeletal muscle displayed Ca2+-dependent binding to liposomes containing phosphatidylinositol 4,5-bisphosphate and phosphatidylcholine. Binding saturated at 1/20 molar ratio phosphatidylinositol 4,5-bisphosphate/phosphatidylcholine and was optimal at free [Ca2+] > or = 20 mM. Extraction of Annexin VI from triads did not affect the generation of phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate, or phosphatidic acid by endogenous lipid kinases, suggesting that despite its capacity to bind to negatively charged phospholipids, Annexin VI does not affect the kinase activities responsible for their generation.

  2. Evolution of the isoprene biosynthetic pathway in kudzu.

    PubMed

    Sharkey, Thomas D; Yeh, Sansun; Wiberley, Amy E; Falbel, Tanya G; Gong, Deming; Fernandez, Donna E

    2005-02-01

    Isoprene synthase converts dimethylallyl diphosphate, derived from the methylerythritol 4-phosphate (MEP) pathway, to isoprene. Isoprene is made by some plants in substantial amounts, which affects atmospheric chemistry, while other plants make no isoprene. As part of our long-term study of isoprene synthesis, the genetics of the isoprene biosynthetic pathway of the isoprene emitter, kudzu (Pueraria montana), was compared with similar genes in Arabidopsis (Arabidopsis thaliana), which does not make isoprene. The MEP pathway genes in kudzu were similar to the corresponding Arabidopsis genes. Isoprene synthase genes of kudzu and aspen (Populus tremuloides) were cloned to compare their divergence with the divergence seen in MEP pathway genes. Phylogenetic analysis of the terpene synthase gene family indicated that isoprene synthases are either within the monoterpene synthase clade or sister to it. In Arabidopsis, the gene most similar to isoprene synthase is a myrcene/ocimene (acyclic monoterpenes) synthase. Two phenylalanine residues found exclusively in isoprene synthases make the active site smaller than other terpene synthase enzymes, possibly conferring specificity for the five-carbon substrate rather than precursors of the larger isoprenoids. Expression of the kudzu isoprene synthase gene in Arabidopsis caused Arabidopsis to emit isoprene, indicating that whether or not a plant emits isoprene depends on whether or not it has a terpene synthase capable of using dimethylallyl diphosphate.

  3. Structural analysis of substrate-mimicking inhibitors in complex with Neisseria meningitidis 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase - The importance of accommodating the active site water.

    PubMed

    Heyes, Logan C; Reichau, Sebastian; Cross, Penelope J; Jameson, Geoffrey B; Parker, Emily J

    2014-12-01

    3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first committed step of the shikimate pathway, which produces the aromatic amino acids as well as many other aromatic metabolites. DAH7PS catalyses an aldol-like reaction between phosphoenolpyruvate and erythrose 4-phosphate. Three phosphoenolpyruvate mimics, (R)-phospholactate, (S)-phospholactate and vinyl phosphonate [(E)-2-methyl-3-phosphonoacrylate], were found to competitively inhibit DAH7PS from Neisseria meningitidis, which is the pathogen responsible for bacterial meningitis. The most potent inhibitor was the vinyl phosphonate with a Ki value of 3.9±0.4μM. We report for the first time crystal structures of these compounds bound in the active site of a DAH7PS enzyme which reveals that the inhibitors bind to the active site of the enzyme in binding modes that mimic those of the predicted oxocarbenium and tetrahedral intermediates of the enzyme-catalysed reaction. Furthermore, the inhibitors accommodate the binding of a key active site water molecule. Together, these observations provide strong evidence that this active site water participates directly in the DAH7PS reaction, enabling the facial selectivity of the enzyme-catalysed reaction sequence to be delineated.

  4. Mineral induced formation of sugar phosphates

    NASA Technical Reports Server (NTRS)

    Pitsch, S.; Eschenmoser, A.; Gedulin, B.; Hui, S.; Arrhenius, G.

    1995-01-01

    Glycolaldehyde phosphate, sorbed from highly dilute, weakly alkaline solution into the interlayer of common expanding sheet structure metal hydroxide minerals, condenses extensively to racemic aldotetrose-2, 4-diphophates, and aldohexose-2, 4, 6-triphosphates. The reaction proceeds mainly through racemic erythrose-2, 4-phosphate, and terminates with a large fraction of racemic altrose-2, 4, 6-phosphate. In the absence of an inductive mineral phase, no detectable homogeneous reaction takes place in the concentration- and pH range used. The reactant glycolaldehyde phosphate is practically completely sorbed within an hour from solutions with concentrations as low as 50 micron; the half-time for conversion to hexose phosphates is of the order of two days at room temperature and pH 9.5. Total production of sugar phosphates in the mineral interlayer is largely independent of the glycolaldehyde phosphate concentration in the external solution, but is determined by the total amount of GAP offered for sorption up to the capacity of the mineral. In the presence of equimolar amounts of rac-glyceraldehyde-2-phosphate, but under otherwise similar conditions, aldopentose-2, 4, -diphosphates also form, but only as a small fraction of the hexose-2, 4, 6-phosphates.

  5. 2C-Methyl- D- erythritol 2,4-cyclodiphosphate synthase from Stevia rebaudiana Bertoni is a functional gene.

    PubMed

    Kumar, Hitesh; Singh, Kashmir; Kumar, Sanjay

    2012-12-01

    Stevia [Stevia rebaudiana (Bertoni)] is a perennial herb which accumulates sweet diterpenoid steviol glycosides (SGs) in its leaf tissue. SGs are synthesized by 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. Of the various enzymes of the MEP pathway, 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MDS) (encoded by MDS) catalyzes the cyclization of 4-(cytidine 5' diphospho)-2C-methyl-D-erythritol 2-phosphate into 2C-methyl-D-erythritol 2,4-cyclodiphosphate. Complementation of the MDS knockout mutant strain of Escherichia coli, EB370 with putative MDS of stevia (SrMDS) rescued the lethal mutant, suggesting SrMDS to be a functional gene. Experiments conducted in plant growth chamber and in the field suggested SrMDS to be a light regulated gene. Indole 3-acetic acid (IAA; 50, 100 μM) down-regulated the expression of SrMDS at 4 h of the treatment, whereas, abscisic acid did not modulate its expression. A high expression of SrMDS was observed during the light hours of the day as compared to the dark hours. The present work established functionality of SrMDS and showed the role of light and IAA in regulating expression of SrMDS.

  6. A functional (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase exhibits diurnal regulation of expression in Stevia rebaudiana (Bertoni).

    PubMed

    Kumar, Hitesh; Kumar, Sanjay

    2013-09-15

    The leaves of stevia [Stevia rebaudiana (Bertoni)] are a rich source of steviol glycosides that are used as non-calorific sweetener in many countries around the world. Steviol moiety of steviol glycosides is synthesized via plastidial 2C-methyl-D-erythritol 4-phosphate pathway, where (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is the key enzyme. HDR catalyzes the simultaneous conversion of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate into five carbon isoprenoid units, isopentenyl diphosphate and dimethylallyl diphosphate. Stevia HDR (SrHDR) successfully rescued HDR lethal mutant strain MG1655 ara<>ispH upon genetic complementation, suggesting SrHDR to encode a functional protein. The gene exhibited diurnal variation in expression. To identify the possible regulatory elements, upstream region of the gene was cloned and putative cis-acting elements were detected by in silico analysis. Electrophoretic mobility shift assay, using a putative light responsive element GATA showed the binding of nuclear proteins (NP) isolated from leaves during light period of the day, but not with the NP from leaves during the dark period. Data suggested the involvement of GATA box in light mediated gene regulation of SrHDR in stevia.

  7. The Polycystin-1, Lipoxygenase, and α-Toxin Domain Regulates Polycystin-1 Trafficking.

    PubMed

    Xu, Yaoxian; Streets, Andrew J; Hounslow, Andrea M; Tran, Uyen; Jean-Alphonse, Frederic; Needham, Andrew J; Vilardaga, Jean-Pierre; Wessely, Oliver; Williamson, Michael P; Ong, Albert C M

    2016-04-01

    Mutations in polycystin-1 (PC1) give rise to autosomal dominant polycystic kidney disease, an important and common cause of kidney failure. Despite its medical importance, the function of PC1 remains poorly understood. Here, we investigated the role of the intracellular polycystin-1, lipoxygenase, and α-toxin (PLAT) signature domain of PC1 using nuclear magnetic resonance, biochemical, cellular, and in vivo functional approaches. We found that the PLAT domain targets PC1 to the plasma membrane in polarized epithelial cells by a mechanism involving the selective binding of the PLAT domain to phosphatidylserine and L-α-phosphatidylinositol-4-phosphate (PI4P) enriched in the plasma membrane. This process is regulated by protein kinase A phosphorylation of the PLAT domain, which reduces PI4P binding and recruits β-arrestins and the clathrin adaptor AP2 to trigger PC1 internalization. Our results reveal a physiological role for the PC1-PLAT domain in renal epithelial cells and suggest that phosphorylation-dependent internalization of PC1 is closely linked to its function in renal development and homeostasis.

  8. Overexpression and Suppression of Artemisia annua 4-Hydroxy-3-Methylbut-2-enyl Diphosphate Reductase 1 Gene (AaHDR1) Differentially Regulate Artemisinin and Terpenoid Biosynthesis

    PubMed Central

    Ma, Dongming; Li, Gui; Zhu, Yue; Xie, De-Yu

    2017-01-01

    4-Hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) catalyzes the last step of the 2-C-methyl-D-erythritol 4- phosphate (MEP) pathway to synthesize isopentenyl pyrophosphate (IPP) and dimethylallyl diphosphate (DMAPP). To date, little is known regarding effects of an increase or a decrease of a HDR expression on terpenoid and other metabolite profiles in plants. In our study, an Artemisia annua HDR cDNA (namely AaHDR1) was cloned from leaves. Expression profiling showed that it was highly expressed in leaves, roots, stems, and flowers with different levels. Green florescence protein fusion and confocal microscope analyses showed that AaHDR1 was localized in chloroplasts. The overexpression of AaHDR1 increased contents of artemisinin, arteannuin B and other sesquiterpenes, and multiple monoterpenes. By contrast, the suppression of AaHDR1 by anti-sense led to opposite results. In addition, an untargeted metabolic profiling showed that the overexpression and suppression altered non-polar metabolite profiles. In conclusion, the overexpression and suppression of AaHDR1 protein level in plastids differentially affect artemisinin and other terpenoid biosynthesis, and alter non-polar metabolite profiles of A. annua. Particularly, its overexpression leading to the increase of artemisinin production is informative to future metabolic engineering of this antimalarial medicine. PMID:28197158

  9. Influence of cyclic nucleotides (cAMP) on inositol phospholipid (InsPL) metabolism in cultured mesangial (MS) cells

    SciTech Connect

    Troyer, D.A.; Venkatachalam, M.A.; Bonventre, J.V.; Kreisberg, J.I.

    1986-03-01

    Elevation of cAMP inhibits hormone-induced contraction of MS cells, and in other cell types, reduces stimulated InsPL metabolism. The authors found that neither isobutylmethylxanthine (MIX, 0.5 mM), which increases MS cell cAMP levels 4-fold, nor forskolin (100 ..mu..M) altered vasopressin (VP, 10 nM) induced release of /sup 3/H-inositol trisphosphate from prelabelled MS cells. Also, maneuvers which elevated cAMP did not block the VP-induced rise of intracellular calcium as measured by quin-2. Further, neither MIX nor isoproterenol affected the stimulation of glycolysis by VP as measured by lactic acid production. MIX diminished VP stimulated /sup 32/P orthophosphate (/sup 32/P) incorporation into phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol 4-phosphate and phosphatidylinositol. The /sup 32/P content in phosphoinositides of cells treated with MIX and VP was 65% of that in cells treated with VP only. However, the authors found that the specific activity of /sup 32/P in ATP in the presence of MIX + VP was 74% of that with VP alone. Thus, the apparent suppression of /sup 32/P incorporation due to MIX was attributable to a concurrent diminution of the specific activity of /sup 32/P in ATP. The authors conclude that increases of cAMP interfere with contraction distal to PIP/sub 2/ hydrolysis, inositol phosphate release, calcium mobilization, and enhancement of glycolysis.

  10. A Geranylfarnesyl Diphosphate Synthase Provides the Precursor for Sesterterpenoid (C25) Formation in the Glandular Trichomes of the Mint Species Leucosceptrum canum

    PubMed Central

    Luo, Shi-Hong; Schmidt, Axel; Sun, Gui-Ling; Kuang, Ce; Yang, Min-Jie; Jing, Shu-Xi; Li, Chun-Huan

    2016-01-01

    Plant sesterterpenoids, an important class of terpenoids, are widely distributed in various plants, including food crops. However, little is known about their biosynthesis. Here, we cloned and functionally characterized a plant geranylfarnesyl diphosphate synthase (Lc-GFDPS), the enzyme producing the C25 prenyl diphosphate precursor to all sesterterpenoids, from the glandular trichomes of the woody plant Leucosceptrum canum. GFDPS catalyzed the formation of GFDP after expression in Escherichia coli. Overexpressing GFDPS in Arabidopsis thaliana also gave an extract catalyzing GFDP formation. GFDPS was strongly expressed in glandular trichomes, and its transcript profile was completely in accordance with the sesterterpenoid accumulation pattern. GFDPS is localized to the plastids, and inhibitor studies indicated its use of isoprenyl diphosphate substrates supplied by the 2-C-methyl-d-erythritol 4-phosphate pathway. Application of a jasmonate defense hormone induced GFDPS transcript and sesterterpenoid accumulation, while reducing feeding and growth of the generalist insect Spodoptera exigua, suggesting that these C25 terpenoids play a defensive role. Phylogenetic analysis suggested that GFDPS probably evolved from plant geranylgeranyl diphosphate synthase under the influence of positive selection. The isolation of GFDPS provides a model for investigating sesterterpenoid formation in other species and a tool for manipulating the formation of this group in plants and other organisms. PMID:26941091

  11. In vivo and in vitro evaluation of a microencapsulated narcotic antagonist.

    PubMed

    Mason, N; Thies, C; Cicero, T J

    1976-06-01

    Injectable microcapsules containing 75% (w/w) cyclazocine, a narcotic antagonist, were prepared with dl-poly(lactic acid) as the coating material. Capsule fractions falling between 105 and 295 mum released about 90% of their cyclazocine in 8 days of rotating-bottle extraction at 37 degrees in pH 7.4 phosphate buffer. Although larger capsules released the drug somewhat more slowly, all capsules released cyclazocine far more rapidly than an ideal capsule should. This rapid release is attributed to macroscopic defects located in the capsule walls. The ability of the capsules to block the action of morphine in vivo was assessed by injection of a sesame seed oil suspension into Holtzman rats. A hot-plate test procedure was used to evaluate animal behavior. Capsule doses of 100-250 mg/kg to rats caused significant antagonism of morphine's analgesic effect for 14 days after injection. By Day 17, no antagonism occurred, indicating that the capsules completely released the drug in vivo between 14 and 17 days after injection.

  12. Phenotypic mutants of the intracellular actinomycete Rhodococcus equi created by in vivo Himar1 transposon mutagenesis.

    PubMed

    Ashour, Joseph; Hondalus, Mary K

    2003-04-01

    Rhodococcus equi is a facultative intracellular opportunistic pathogen of immunocompromised people and a major cause of pneumonia in young horses. An effective live attenuated vaccine would be extremely useful in the prevention of R. equi disease in horses. Toward that end, we have developed an efficient transposon mutagenesis system that makes use of a Himar1 minitransposon delivered by a conditionally replicating plasmid for construction of R. equi mutants. We show that Himar1 transposition in R. equi is random and needs no apparent consensus sequence beyond the required TA dinucleotide. The diversity of the transposon library was demonstrated by the ease with which we were able to screen for auxotrophs and mutants with pigmentation and capsular phenotypes. One of the pigmentation mutants contained an insertion in a gene encoding phytoene desaturase, an enzyme of carotenoid biosynthesis, the pathway necessary for production of the characteristic salmon color of R. equi. We identified an auxotrophic mutant with a transposon insertion in the gene encoding a putative dual-functioning GTP cyclohydrolase II-3,4-dihydroxy-2-butanone-4-phosphate synthase, an enzyme essential for riboflavin biosynthesis. This mutant cannot grow in minimal medium in the absence of riboflavin supplementation. Experimental murine infection studies showed that, in contrast to wild-type R. equi, the riboflavin-requiring mutant is attenuated because it is unable to replicate in vivo. The mutagenesis methodology we have developed will allow the characterization of R. equi virulence mechanisms and the creation of other attenuated strains with vaccine potential.

  13. Deoxyxylulose 5-phosphate reductoisomerase is not a rate-determining enzyme for essential oil production in spike lavender.

    PubMed

    Mendoza-Poudereux, Isabel; Muñoz-Bertomeu, Jesús; Arrillaga, Isabel; Segura, Juan

    2014-11-01

    Spike lavender (Lavandula latifolia) is an economically important aromatic plant producing essential oils, whose components (mostly monoterpenes) are mainly synthesized through the plastidial methylerythritol 4-phosphate (MEP) pathway. 1-Deoxy-D-xylulose-5-phosphate (DXP) synthase (DXS), that catalyzes the first step of the MEP pathway, plays a crucial role in monoterpene precursors biosynthesis in spike lavender. To date, however, it is not known whether the DXP reductoisomerase (DXR), that catalyzes the conversion of DXP into MEP, is also a rate-limiting enzyme for the biosynthesis of monoterpenes in spike lavender. To investigate it, we generated transgenic spike lavender plants constitutively expressing the Arabidopsis thaliana DXR gene. Although two out of the seven transgenic T0 plants analyzed accumulated more essential oils than the controls, this is hardly imputable to the DXR transgene effect since a clear correlation between transcript accumulation and monoterpene production could not be established. Furthermore, these increased essential oil phenotypes were not maintained in their respective T1 progenies. Similar results were obtained when total chlorophyll and carotenoid content in both T0 transgenic plants and their progenies were analyzed. Our results then demonstrate that DXR enzyme does not play a crucial role in the synthesis of plastidial monoterpene precursors, suggesting that the control flux of the MEP pathway in spike lavender is primarily exerted by the DXS enzyme.

  14. Enhanced levels of S-linalool by metabolic engineering of the terpenoid pathway in spike lavender leaves.

    PubMed

    Mendoza-Poudereux, Isabel; Muñoz-Bertomeu, Jesús; Navarro, Alicia; Arrillaga, Isabel; Segura, Juan

    2014-05-01

    Transgenic Lavandula latifolia plants overexpressing the linalool synthase (LIS) gene from Clarkia breweri, encoding the LIS enzyme that catalyzes the synthesis of linalool were generated. Most of these plants increased significantly their linalool content as compared to controls, especially in the youngest leaves, where a linalool increase up to a 1000% was observed. The phenotype of increased linalool content observed in young leaves was maintained in those T1 progenies that inherit the LIS transgene, although this phenotype was less evident in the flower essential oil. Cross-pollination of transgenic spike lavender plants allowed the generation of double transgenic plants containing the DXS (1-deoxy-d-xylulose-5-P synthase), coding for the first enzyme of the methyl-d-erythritol-4-phosphate pathway, and LIS genes. Both essential oil yield and linalool content in double DXS-LIS transgenic plants were lower than that of their parentals, which could be due to co-suppression effects linked to the structures of the constructs used.

  15. Acid inactivation of and incorporation of phosphate into alkaline phosphatase from Escherichia coli

    PubMed Central

    Pigretti, M. M.; Milstein, C.

    1965-01-01

    1. Alkaline phosphatase of Escherichia coli undergoes below pH 6·0 a reversible acid inactivation that has been studied and related to the extent of uptake of inorganic phosphate occurring below pH 6·0. 2. The rate of inactivation is rapid in the first few minutes but later it decreases markedly. Temperature, pH, composition of buffer and other factors have an important effect on the inactivation. 3. About 60% of the activity lost at pH values above 3·5 is rapidly recovered when the enzyme is taken back to pH 8·0, independently (within certain limits) of the extent of the inactivation. 4. Phosphate and Zn2+, although very good protectors of the inactivation by acid, are not by themselves able to reverse the acid inactivation. 5. Inorganic phosphate seems not to be incorporated into the acid-inactivated enzyme. 6. Incorporation of more than one mole of phosphate/mole of enzyme has been obtained, but the phosphate residues seem to be incorporated to serine residues with a common sequence, suggesting two identical active serine residues/molecule of active enzyme. ImagesFig. 3.Fig. 4. PMID:14342215

  16. Overexpressing 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase (HMGR) in the Lactococcal Mevalonate Pathway for Heterologous Plant Sesquiterpene Production

    PubMed Central

    Song, Adelene Ai-Lian; Abdullah, Janna Ong; Abdullah, Mohd. Puad; Shafee, Norazizah; Othman, Roohaida; Tan, Ee-Fun; Noor, Normah Mohd.; Raha, Abdul Rahim

    2012-01-01

    Isoprenoids are a large and diverse group of metabolites with interesting properties such as flavour, fragrance and therapeutic properties. They are produced via two pathways, the mevalonate pathway or the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. While plants are the richest source of isoprenoids, they are not the most efficient producers. Escherichia coli and yeasts have been extensively studied as heterologous hosts for plant isoprenoids production. In the current study, we describe the usage of the food grade Lactococcus lactis as a potential heterologous host for the production of sesquiterpenes from a local herbaceous Malaysian plant, Persicaria minor (synonym Polygonum minus). A sesquiterpene synthase gene from P. minor was successfully cloned and expressed in L. lactis. The expressed protein was identified to be a β-sesquiphellandrene synthase as it was demonstrated to be functional in producing β-sesquiphellandrene at 85.4% of the total sesquiterpenes produced based on in vitro enzymatic assays. The recombinant L. lactis strain developed in this study was also capable of producing β-sesquiphellandrene in vivo without exogenous substrates supplementation. In addition, overexpression of the strain’s endogenous 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR), an established rate-limiting enzyme in the eukaryotic mevalonate pathway, increased the production level of β-sesquiphellandrene by 1.25–1.60 fold. The highest amount achieved was 33 nM at 2 h post-induction. PMID:23300671

  17. Oxysterol-binding proteins: sterol and phosphoinositide sensors coordinating transport, signaling and metabolism.

    PubMed

    Olkkonen, Vesa M; Li, Shiqian

    2013-10-01

    Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute a family of sterol and phosphoinositide binding proteins conserved in eukaryotes. The mechanisms of ORP function have remained incompletely understood. However, several ORPs are present at membrane contact sites and control the activity of enzymatic effectors or assembly of protein complexes, with impacts on signaling, vesicle transport, and lipid metabolism. An increasing number of protein interaction partners of ORPs have been identified, providing clues of their involvement in multiple aspects of cell regulation. The functions assigned for mammalian ORPs include coordination of sterol and sphingolipid metabolism and mitogenic signaling (OSBP), control of ER-late endosome (LE) contacts and LE motility (ORP1L), neutral lipid metabolism (ORP2), cell adhesion (ORP3), cholesterol eggress from LE (ORP5), macrophage lipid homeostasis, migration and high-density lipoprotein metabolism (ORP8), apolipoprotein B-100 secretion (ORP10), and adipogenesis (ORP11). The anti-proliferative ORPphilin compounds target OSBP and ORP4, revealing a function of ORPs in cell proliferation and survival. The Saccharomyces cerevisiae OSBP homologue (Osh) proteins execute multifaceted functions in sterol and sphingolipid homeostasis, post-Golgi vesicle transport, as well as phosphatidylinositol-4-phosphate and target of rapamycin complex 1 (TORC1) signaling. These observations identify ORPs as coordinators of lipid signals with an unforeseen variety of cellular processes.

  18. Electrochemical behavior of an antiviral drug acyclovir at fullerene-C(60)-modified glassy carbon electrode.

    PubMed

    Shetti, Nagaraj P; Malode, Shweta J; Nandibewoor, Sharanappa T

    2012-12-01

    Electrochemical oxidation of acyclovir at fullerene-C(60)-modified glassy carbon electrode has been investigated using cyclic and differential pulse voltammetry. In pH 7.4 phosphate buffer, acyclovir showed an irreversible oxidation peak at about 0.96V. The cyclic voltammetric results showed that fullerene-C(60)-modified glassy carbon electrode can remarkably enhance electrocatalytic activity towards the oxidation of acyclovir. The electrocatalytic behavior was further exploited as a sensitive detection scheme for the acyclovir determination by differential pulse voltammetry. Effects of anodic peak potential (E(p)/V), anodic peak current (I(p)/μA) and heterogeneous rate constant (k(0)) have been discussed. Under optimized conditions, the concentration range and detection limit were 9.0×10(-8) to 6.0×10(-6)M and 1.48×10(-8)M, respectively. The proposed method was applied to acyclovir determination in pharmaceutical samples and human biological fluids such as urine and blood plasma as a real sample. This method can also be employed in quality control and routine determination of drugs in pharmaceutical formulations.

  19. Genetic structure and regulation of isoprene synthase in Poplar (Populus spp.).

    PubMed

    Vickers, Claudia E; Possell, Malcolm; Nicholas Hewitt, C; Mullineaux, Philip M

    2010-07-01

    Isoprene is a volatile 5-carbon hydrocarbon derived from the chloroplastic methylerythritol 2-C-methyl-D: -erythritol 4-phosphate isoprenoid pathway. In plants, isoprene emission is controlled by the enzyme isoprene synthase; however, there is still relatively little known about the genetics and regulation of this enzyme. Isoprene synthase gene structure was analysed in three poplar species. It was found that genes encoding stromal isoprene synthase exist as a small gene family, the members of which encode virtually identical proteins and are differentially regulated. Accumulation of isoprene synthase protein is developmentally regulated, but does not differ between sun and shade leaves and does not increase when heat stress is applied. Our data suggest that, in mature leaves, isoprene emission rates are primarily determined by substrate (dimethylallyl diphosphate, DMADP) availability. In immature leaves, where isoprene synthase levels are variable, emission levels are also influenced by the amount of isoprene synthase protein. No thylakoid isoforms could be identified in Populus alba or in Salix babylonica. Together, these data show that control of isoprene emission at the genetic level is far more complicated than previously assumed.

  20. Cross-talk between the cytosolic mevalonate and the plastidial methylerythritol phosphate pathways in tobacco bright yellow-2 cells.

    PubMed

    Hemmerlin, Andréa; Hoeffler, Jean-François; Meyer, Odile; Tritsch, Denis; Kagan, Isabelle A; Grosdemange-Billiard, Catherine; Rohmer, Michel; Bach, Thomas J

    2003-07-18

    In plants, two pathways are utilized for the synthesis of isopentenyl diphosphate, the universal precursor for isoprenoid biosynthesis. The key enzyme of the cytoplasmic mevalonic acid (MVA) pathway is 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR). Treatment of Tobacco Bright Yellow-2 (TBY-2) cells by the HMGR-specific inhibitor mevinolin led to growth reduction and induction of apparent HMGR activity, in parallel to an increase in protein representing two HMGR isozymes. Maximum induction was observed at 24 h. 1-Deoxy-d-xylulose (DX), the dephosphorylated first precursor of the plastidial 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, complemented growth inhibition by mevinolin in the low millimolar concentration range. Furthermore, DX partially re-established feedback repression of mevinolin-induced HMGR activity. Incorporation studies with [1,1,1,4-2H4]DX showed that sterols, normally derived from MVA, in the presence of mevinolin are synthesized via the MEP pathway. Fosmidomycin, an inhibitor of 1-deoxy-d-xylulose-5-phosphate reductoisomerase, the second enzyme of the MEP pathway, was utilized to study the reverse complementation. Growth inhibition by fosmidomycin of TBY-2 cells could be partially overcome by MVA. Chemical complementation was further substantiated by incorporation of [2-13C]MVA into plastoquinone, representative of plastidial isoprenoids. Best rates of incorporation of exogenous stably labeled precursors were observed in the presence of both inhibitors, thereby avoiding internal isotope dilution.

  1. Arf6 controls retromer traffic and intracellular cholesterol distribution via a phosphoinositide-based mechanism

    PubMed Central

    Marquer, Catherine; Tian, Huasong; Yi, Julie; Bastien, Jayson; Dall'Armi, Claudia; Yang-Klingler, YoungJoo; Zhou, Bowen; Chan, Robin Barry; Di Paolo, Gilbert

    2016-01-01

    Small GTPases play a critical role in membrane traffic. Among them, Arf6 mediates transport to and from the plasma membrane, as well as phosphoinositide signalling and cholesterol homeostasis. Here we delineate the molecular basis for the link between Arf6 and cholesterol homeostasis using an inducible knockout (KO) model of mouse embryonic fibroblasts (MEFs). We find that accumulation of free cholesterol in the late endosomes/lysosomes of Arf6 KO MEFs results from mistrafficking of Niemann–Pick type C protein NPC2, a cargo of the cation-independent mannose-6-phosphate receptor (CI-M6PR). This is caused by a selective increase in an endosomal pool of phosphatidylinositol-4-phosphate (PI4P) and a perturbation of retromer, which controls the retrograde transport of CI-M6PR via sorting nexins, including the PI4P effector SNX6. Finally, reducing PI4P levels in KO MEFs through independent mechanisms rescues aberrant retromer tubulation and cholesterol mistrafficking. Our study highlights a phosphoinositide-based mechanism for control of cholesterol distribution via retromer. PMID:27336679

  2. Crystallization and preliminary X-ray analysis of 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE) from Mycobacterium tuberculosis.

    PubMed

    Shan, Shan; Chen, Xuehui

    2011-07-01

    The 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE) from Mycobacterium tuberculosis, an enzyme from the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, is crucial and essential for the survival of this pathogenic bacterium. IspE catalyzes the conversion of 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME) to 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate (CDP-ME2P) in an ATP-dependent manner. Solving the crystal structure of M. tuberculosis IspE will shed light on its structural details and mechanism of action and may provide the basis for the future design of drugs for the treatment of multidrug-resistant and extremely drug-resistant M. tuberculosis strains. Recombinant M. tuberculosis IspE was crystallized at 291 K using NaCl or Li2SO4 as a precipitant. A 2.1 Å resolution native data set was collected from a single flash-cooled crystal (100 K) belonging to space group P2(1)2(1)2(1), with unit-cell parameters a=52.5, b=72.3, c=107.3 Å. One molecule was assumed per asymmetric unit, which gives a Matthews coefficient of 3.4 Å3 Da(-1) with 63% solvent content.

  3. Design of novel ligands of CDP-methylerythritol kinase by mimicking direct protein-protein and solvent-mediated interactions.

    PubMed

    Giménez-Oya, Victor; Villacañas, Oscar; Obiol-Pardo, Cristian; Antolin-Llovera, Meritxell; Rubio-Martinez, Jaime; Imperial, Santiago

    2011-01-01

    The methylerythritol 4-phosphate (MEP) pathway for the biosynthesis of the isoprenoid universal building blocks (isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP)) is present in most of human pathogens and is absent in animals, turning it into a promising therapeutic druggable pathway. Two different strategies, a pharmacophore-directed virtual screening and a protein-protein interaction (PPI)-mimicking cyclic peptide were used to search for compounds that bind to the PPI surface of the 4-(cytidine 5-diphospho)-2C-methyl-D-erythritol kinase (CMK), which catalyzes the fourth step of the MEP pathway. A significant part of the pharmacophore hypothesis used in this study was designed by mimicking water-mediated PPI relevant in the CMK homodimer complex stabilization. After database search and with the aid of docking and molecular dynamics (MD) simulations, a 7H-furo[3,2-g]chromen-7-one derivative and a cyclic peptide were chosen as candidates to be ligands of CMK. Their binding affinities were measured using surface plasmon resonance (SPR) technology.

  4. A Chemical Rescue Screen Identifies a Plasmodium falciparum Apicoplast Inhibitor Targeting MEP Isoprenoid Precursor Biosynthesis

    PubMed Central

    Wu, Wesley; Herrera, Zachary; Ebert, Danny; Baska, Katie; Cho, Seok H.

    2014-01-01

    The apicoplast is an essential plastid organelle found in Plasmodium parasites which contains several clinically validated antimalarial-drug targets. A chemical rescue screen identified MMV-08138 from the “Malaria Box” library of growth-inhibitory antimalarial compounds as having specific activity against the apicoplast. MMV-08138 inhibition of blood-stage Plasmodium falciparum growth is stereospecific and potent, with the most active diastereomer demonstrating a 50% effective concentration (EC50) of 110 nM. Whole-genome sequencing of 3 drug-resistant parasite populations from two independent selections revealed E688Q and L244I mutations in P. falciparum IspD, an enzyme in the MEP (methyl-d-erythritol-4-phosphate) isoprenoid precursor biosynthesis pathway in the apicoplast. The active diastereomer of MMV-08138 directly inhibited PfIspD activity in vitro with a 50% inhibitory concentration (IC50) of 7.0 nM. MMV-08138 is the first PfIspD inhibitor to be identified and, together with heterologously expressed PfIspD, provides the foundation for further development of this promising antimalarial drug candidate lead. Furthermore, this report validates the use of the apicoplast chemical rescue screen coupled with target elucidation as a discovery tool to identify specific apicoplast-targeting compounds with new mechanisms of action. PMID:25367906

  5. Differential Subplastidial Localization and Turnover of Enzymes Involved in Isoprenoid Biosynthesis in Chloroplasts

    PubMed Central

    Perello, Catalina; Llamas, Ernesto; Burlat, Vincent; Ortiz-Alcaide, Miriam; Phillips, Michael A.; Pulido, Pablo; Rodriguez-Concepcion, Manuel

    2016-01-01

    Plastidial isoprenoids are a diverse group of metabolites with roles in photosynthesis, growth regulation, and interaction with the environment. The methylerythritol 4-phosphate (MEP) pathway produces the metabolic precursors of all types of plastidial isoprenoids. Proteomics studies in Arabidopsis thaliana have shown that all the enzymes of the MEP pathway are localized in the plastid stroma. However, immunoblot analysis of chloroplast subfractions showed that the first two enzymes of the pathway, deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR), can also be found in non-stromal fractions. Both transient and stable expression of GFP-tagged DXS and DXR proteins confirmed the presence of the fusion proteins in distinct subplastidial compartments. In particular, DXR-GFP was found to accumulate in relatively large vesicles that could eventually be released from chloroplasts, presumably to be degraded by an autophagy-independent process. Together, we propose that protein-specific mechanisms control the localization and turnover of the first two enzymes of the MEP pathway in Arabidopsis chloroplasts. PMID:26919668

  6. Absence of substrate channeling between active sites in the Agrobacterium tumefaciens IspDF and IspE enzymes of the methyl erythritol phosphate pathway.

    PubMed

    Lherbet, Christian; Pojer, Florence; Richard, Stéphane B; Noel, Joseph P; Poulter, C D

    2006-03-21

    The conversion of 2-C-methyl-d-erythritol 4-phosphate (MEP) to 2-C-methyl-d-erythritol 2,4-cyclodiphosphate (cMEDP) in the MEP entry into the isoprenoid biosynthetic pathway occurs in three consecutive steps catalyzed by the IspD, IspE, and IspF enzymes, respectively. In Agrobacterium tumefaciens the ispD and ispF genes are fused to encode a bifunctional enzyme that catalyzes the first (synthesis of 4-diphosphocytidyl-2-C-methyl d-erythritol) and third (synthesis of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate) steps. Sedimentation velocity experiments indicate that the bifunctional IspDF enzyme and the IspE protein associate in solution, raising the possibility of substrate channeling among the active sites in these two proteins. Kinetic evidence for substrate channeling was sought by measuring the time courses for product formation during incubations of MEP, CTP, and ATP with the IspDF and IspE proteins with and without an excess of the inactive IspE(D152A) mutant in the presence or absence of 30% (v/v) glycerol. The time dependencies indicate that the enzyme-generated intermediates are not transferred from the IspD active site in IspDF to the active site of IspE or from the active site in IspE to the active site of the IspF module of IspDF.

  7. Molecular Mechanism of Action of Antimalarial Benzoisothiazolones: Species-Selective Inhibitors of the Plasmodium spp. MEP Pathway enzyme, IspD

    PubMed Central

    Price, Kathryn E.; Armstrong, Christopher M.; Imlay, Leah S.; Hodge, Dana M.; Pidathala, C.; Roberts, Natalie J.; Park, Jooyoung; Mikati, Marwa; Sharma, Raman; Lawrenson, Alexandre S.; Tolia, Niraj H.; Berry, Neil G.; O’Neill, Paul M.; John, Audrey R. Odom

    2016-01-01

    The methylerythritol phosphate (MEP) pathway is an essential metabolic pathway found in malaria parasites, but absent in mammals, making it a highly attractive target for the discovery of novel and selective antimalarial therapies. Using high-throughput screening, we have identified 2-phenyl benzo[d]isothiazol-3(2H)-ones as species-selective inhibitors of Plasmodium spp. 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase (IspD), the third catalytic enzyme of the MEP pathway. 2-Phenyl benzo[d]isothiazol-3(2H)-ones display nanomolar inhibitory activity against P. falciparum and P. vivax IspD and prevent the growth of P. falciparum in culture, with EC50 values below 400 nM. In silico modeling, along with enzymatic, genetic and crystallographic studies, have established a mechanism-of-action involving initial non-covalent recognition of inhibitors at the IspD binding site, followed by disulfide bond formation through attack of an active site cysteine residue on the benzo[d]isothiazol-3(2H)-one core. The species-selective inhibitory activity of these small molecules against Plasmodium spp. IspD and cultured parasites suggests they have potential as lead compounds in the pursuit of novel drugs to treat malaria. PMID:27857147

  8. Organ- and Growing Stage-Specific Expression of Solanesol Biosynthesis Genes in Nicotiana tabacum Reveals Their Association with Solanesol Content.

    PubMed

    Yan, Ning; Zhang, Hongbo; Zhang, Zhongfeng; Shi, John; Timko, Michael P; Du, Yongmei; Liu, Xinmin; Liu, Yanhua

    2016-11-15

    Solanesol is a noncyclic terpene alcohol that is composed of nine isoprene units and mainly accumulates in solanaceous plants, especially tobacco (Nicotiana tabacum L.). In the present study, RNA-seq analyses of tobacco leaves, stems, and roots were used to identify putative solanesol biosynthesis genes. Six 1-deoxy-d-xylulose 5-phosphate synthase (DXS), two 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), two 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase (IspD), four 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase (IspE), two 2-C-methyl-d-erythritol 2,4-cyclo-diphosphate synthase (IspF), four 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (IspG), two 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (IspH), six isopentenyl diphosphate isomerase (IPI), and two solanesyl diphosphate synthase (SPS) candidate genes were identified in the solanesol biosynthetic pathway. Furthermore, the two N. tabacum SPS proteins (NtSPS1 and NtSPS2), which possessed two conserved aspartate-rich DDxxD domains, were highly homologous with SPS enzymes from other solanaceous plant species. In addition, the solanesol contents of three organs and of leaves from four growing stages of tobacco plants corresponded with the distribution of chlorophyll. Our findings provide a comprehensive evaluation of the correlation between the expression of different biosynthesis genes and the accumulation of solanesol, thus providing valuable insight into the regulation of solanesol biosynthesis in tobacco.

  9. A PI4P-driven electrostatic field controls cell membrane identity and signaling in plants

    PubMed Central

    Simon, Mathilde Laetitia Audrey; Platre, Matthieu Pierre; Marquès-Bueno, Maria Mar; Armengot, Laia; Stanislas, Thomas; Bayle, Vincent; Caillaud, Marie-Cécile; Jaillais, Yvon

    2016-01-01

    Many signaling proteins permanently or transiently localize to specific organelles for function. It is well established that certain lipids act as biochemical landmarks to specify compartment identity. However, they also influence membrane biophysical properties, which emerge as important features in specifying cellular territories. Such parameters include the membrane inner surface potential, which varies according to the lipid composition of each organelle. Here, we found that the plant plasma membrane (PM) and the cell plate of dividing cells have a unique electrostatic signature controlled by phosphatidylinositol-4-phosphate (PI4P). Our results further reveal that, contrarily to other eukaryotes, PI4P massively accumulates at the PM, establishing it as a critical hallmark of this membrane in plants. Membrane surface charges control the PM localization and function of the polar auxin transport regulator PINOID, as well as proteins from the BRI1 KINASE INHIBITOR1 (BKI1)/MEMBRANE ASSOCIATED KINASE REGULATORs (MAKRs) family, which are involved in brassinosteroid and receptor-like kinase signaling. We anticipate that this PI4P-driven physical membrane property will control the localization and function of many proteins involved in development, reproduction, immunity and nutrition. PMID:27322096

  10. GOLPH3 drives cell migration by promoting Golgi reorientation and directional trafficking to the leading edge

    PubMed Central

    Xing, Mengke; Peterman, Marshall C.; Davis, Robert L.; Oegema, Karen; Shiau, Andrew K.; Field, Seth J.

    2016-01-01

    The mechanism of directional cell migration remains an important problem, with relevance to cancer invasion and metastasis. GOLPH3 is a common oncogenic driver of human cancers, and is the first oncogene that functions at the Golgi in trafficking to the plasma membrane. Overexpression of GOLPH3 is reported to drive enhanced cell migration. Here we show that the phosphatidylinositol-4-phosphate/GOLPH3/myosin 18A/F-actin pathway that is critical for Golgi–to–plasma membrane trafficking is necessary and limiting for directional cell migration. By linking the Golgi to the actin cytoskeleton, GOLPH3 promotes reorientation of the Golgi toward the leading edge. GOLPH3 also promotes reorientation of lysosomes (but not other organelles) toward the leading edge. However, lysosome function is dispensable for migration and the GOLPH3 dependence of lysosome movement is indirect, via GOLPH3’s effect on the Golgi. By driving reorientation of the Golgi to the leading edge and driving forward trafficking, particularly to the leading edge, overexpression of GOLPH3 drives trafficking to the leading edge of the cell, which is functionally important for directional cell migration. Our identification of a novel pathway for Golgi reorientation controlled by GOLPH3 provides new insight into the mechanism of directional cell migration with important implications for understanding GOLPH3’s role in cancer. PMID:27708138

  11. Molecular insights into the binding of phosphoinositides to the TH domain region of TIPE proteins.

    PubMed

    Antony, Priya; Baby, Bincy; Vijayan, Ranjit

    2016-11-01

    Phosphatidylinositols and their phosphorylated derivatives, phosphoinositides, play a central role in regulating diverse cellular functions. These phospholipids have been shown to interact with the hydrophobic TH domain of the tumor necrosis factor (TNF)-α-induced protein 8 (TIPE) family of proteins. However, the precise mechanism of interaction of these lipids is unclear. Here we report the binding mode and interactions of these phospholipids in the TH domain, as elucidated using molecular docking and simulations. Results indicate that phosphoinositides bind to the TH domain in a similar way by inserting their lipid tails in the hydrophobic cavity. The exposed head group is stabilized by interactions with critical positively charged residues on the surface of these proteins. Further MD simulations confirmed the dynamic stability of these lipids in the TH domain. This computational analysis thus provides insight into the binding mode of phospholipids in the TH domain of the TIPE family of proteins. Graphical abstract A phosphoinositide (phosphatidylinositol 4-phosphate; PtdIns4P) docked to TIPE2.

  12. Competitive inhibition of phosphoglucose isomerase of apple leaves by sorbitol 6-phosphate.

    PubMed

    Zhou, Rui; Cheng, Lailiang

    2008-06-16

    Apple leaf cytosolic phosphoglucose isomerase (PGI, EC 5.3.1.9) was purified to an apparent homogeneity with a specific activity of 2456 units/mg protein, and chloroplastic PGI was partially purified to a specific activity of 72 units/mg protein to characterize their biochemical properties. These two isoforms showed differential responses to heat treatment; incubation at 50 degrees C for 10 min resulted in a complete loss of the chloroplastic PGI activity, whereas the cytosolic PGI only lost 50% of its activity. Apple cytosolic PGI is a dimeric enzyme with a molecular mass of 66 kDa for each monomer. The activity of both isoforms was strongly inhibited by erythrose 4-phosphate (E4P) with a K(i) of 1.2 and 3.0 microM for the cytosolic PGI and chloroplastic PGI, respectively. Sorbitol 6-phosphate (Sor6P), an intermediate in sorbitol biosynthesis, was found to be a competitive inhibitor for both cytosolic and chloroplastic PGIs with a K(i) of 61 and 40 microM, respectively. PGIs from both spinach and tomato leaves were also inhibited by Sor6P in a similar manner. The possible physiological significance of this finding is discussed.

  13. Structure of Yeast OSBP-Related Protein Osh1 Reveals Key Determinants for Lipid Transport and Protein Targeting at the Nucleus-Vacuole Junction.

    PubMed

    Manik, Mohammad Kawsar; Yang, Huiseon; Tong, Junsen; Im, Young Jun

    2017-03-10

    Yeast Osh1 belongs to the oxysterol-binding protein (OSBP) family of proteins and contains multiple targeting modules optimized for lipid transport at the nucleus-vacuole junction (NVJ). The key determinants for NVJ targeting and the role of Osh1 at NVJs have remained elusive because of unknown lipid specificities. In this study, we determined the structures of the ankyrin repeat domain (ANK), and OSBP-related domain (ORD) of Osh1, in complex with Nvj1 and ergosterol, respectively. The Osh1 ANK forms a unique bi-lobed structure that recognizes a cytosolic helical segment of Nvj1. We discovered that Osh1 ORD binds ergosterol and phosphatidylinositol 4-phosphate PI(4)P in a competitive manner, suggesting counter-transport function of the two lipids. Ergosterol is bound to the hydrophobic pocket in a head-down orientation, and the structure of the PI(4)P-binding site in Osh1 is well conserved. Our results suggest that Osh1 performs non-vesicular transport of ergosterol and PI(4)P at the NVJ.

  14. NMR spectral mapping of Lipid A molecular patterns affected by interaction with the innate immune receptor CD14

    SciTech Connect

    Albright, Seth; Agrawal, Prashansa; Jain, Nitin U.

    2009-01-23

    Soluble CD14 (sCD14) is a serum glycoprotein that binds to the Lipid A moiety of lipopolysaccharides (LPS) with high affinity as part of the innate immune response to bacterial endotoxins. In order to investigate structural interactions of Lipid A with sCD14, we have prepared an isotopically labeled form of a fully active and chemically defined endotoxin, Kdo{sub 2}-Lipid A, which allowed us to carry out detailed NMR spectral mapping of this agonist ligand bound to sCD14 and identify for the first time structural regions that are strongly affected during complex formation with sCD14. These map to two adjacent areas comprising the lower portions of the sugar headgroup and upper half of the acyl chains I, III, and V, which are spatially proximal to the 1- and 4'-phosphate ends. Additionally, we have detected for the first time, presence of differential dynamic behavior for the affected resonances, suggesting a likely role for dynamics in the mechanism of Lipid A pattern recognition by sCD14.

  15. Early effects of Escherichia coli endotoxin infusion on vasopressin-stimulated breakdown and metabolism of inositol lipids in rat hepatocytes

    SciTech Connect

    Rodriguez de Turco, E.B.; Spitzer, J.A.

    1988-08-30

    The turnover of vasopressin-stimulated 32P-phosphoinositides and 32P-phosphatidic acid and accumulation of (2-3H)-inositol phosphates were examined in hepatocytes from rats infused i.v. with saline and E. coli endotoxin for 3 hrs. Within 60s of VP stimulation the decrease in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate labeling as well as the increased uptake of 32P into phosphatidic acid were similar in both groups. However, at a later time (300s) the 32P-phosphatidylinositol turnover was greatly decreased concomitantly with a higher labeling of phosphatidic acid. The accumulation of (2-3H)-inositol phosphates in ET-cells was significantly decreased both at 30s and 600s after VP addition. The distribution of (2-3H)-inositol labeling accumulated in the different inositol phosphate fractions over the first 30s of VP stimulation showed a tendency to lower accumulation of inositol trisphosphate, and a significantly lower accumulation of inositol bisphosphate simultaneously with a higher labeling of the inositol tetrakisphosphate fraction. These observations reflect an early effect of ET-infusion on VP-stimulated inositol lipid turnover and on the subsequent metabolism of the released inositol phosphates.

  16. A vibrational spectroscopic study of the anhydrous phosphate mineral sidorenkite Na3Mn(PO4)(CO3)

    NASA Astrophysics Data System (ADS)

    Frost, Ray L.; López, Andrés; Scholz, Ricardo; Belotti, Fernanda Maria; Xi, Yunfei

    2015-02-01

    Sidorenkite is a very rare low-temperature hydrothermal mineral, formed very late in the crystallization of hyperagpaitic pegmatites in a differentiated alkalic massif (Mt. Alluaiv, Kola Peninsula, Russia). Sidorenkite Na3Mn(PO4)(CO3) is a phosphate-carbonate of sodium and manganese. Such a formula with two oxyanions lends itself to vibrational spectroscopy. The sharp Raman band at 959 cm-1 and 1012 cm-1 are assigned to the PO43- stretching modes, whilst the Raman bands at 1044 cm-1 and 1074 cm-1 are attributed to the CO32- stretching modes. It is noted that no Raman bands at around 800 cm-1 for sidorenkite were observed. The infrared spectrum of sidorenkite shows a quite intense band at 868 cm-1 with other resolved component bands at 850 and 862 cm-1. These bands are ascribed to the CO32- out-of-plane bend (ν2) bending mode. The series of Raman bands at 622, 635, 645 and 704 cm-1 are assigned to the ν4 phosphate bending modes. The observation of multiple bands supports the concept of a reduction in symmetry of the carbonate anion from D3h or even C2v.

  17. Differential Contribution of the First Two Enzymes of the MEP Pathway to the Supply of Metabolic Precursors for Carotenoid and Chlorophyll Biosynthesis in Carrot (Daucus carota)

    PubMed Central

    Simpson, Kevin; Quiroz, Luis F.; Rodriguez-Concepción, Manuel; Stange, Claudia R.

    2016-01-01

    Carotenoids and chlorophylls are photosynthetic pigments synthesized in plastids from metabolic precursors provided by the methylerythritol 4-phosphate (MEP) pathway. The first two steps in the MEP pathway are catalyzed by the deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR) enzymes. While DXS has been recently shown to be the main flux-controlling step of the MEP pathway, both DXS and DXR enzymes have been proven to be able to promote an increase in MEP-derived products when overproduced in diverse plant systems. Carrot (Daucus carota) produces photosynthetic pigments (carotenoids and chlorophylls) in leaves and in light-exposed roots, whereas only carotenoids (mainly α- and β-carotene) accumulate in the storage root in darkness. To evaluate whether DXS and DXR activities influence the production of carotenoids and chlorophylls in carrot leaves and roots, the corresponding Arabidopsis thaliana genes were constitutively expressed in transgenic carrot plants. Our results suggest that DXS is limiting for the production of both carotenoids and chlorophylls in roots and leaves, whereas the regulatory role of DXR appeared to be minor. Interestingly, increased levels of DXS (but not of DXR) resulted in higher transcript abundance of endogenous carrot genes encoding phytoene synthase, the main rate-determining enzyme of the carotenoid pathway. These results support a central role for DXS on modulating the production of MEP-derived precursors to synthesize carotenoids and chlorophylls in carrot, confirming the pivotal relevance of this enzyme to engineer healthier, carotenoid-enriched products. PMID:27630663

  18. Recruitment and activation of a lipid kinase by hepatitis C virus NS5A is essential for integrity of the membranous replication compartment.

    PubMed

    Reiss, Simon; Rebhan, Ilka; Backes, Perdita; Romero-Brey, Ines; Erfle, Holger; Matula, Petr; Kaderali, Lars; Poenisch, Marion; Blankenburg, Hagen; Hiet, Marie-Sophie; Longerich, Thomas; Diehl, Sarah; Ramirez, Fidel; Balla, Tamas; Rohr, Karl; Kaul, Artur; Bühler, Sandra; Pepperkok, Rainer; Lengauer, Thomas; Albrecht, Mario; Eils, Roland; Schirmacher, Peter; Lohmann, Volker; Bartenschlager, Ralf

    2011-01-20

    Hepatitis C virus (HCV) is a major causative agent of chronic liver disease in humans. To gain insight into host factor requirements for HCV replication, we performed a siRNA screen of the human kinome and identified 13 different kinases, including phosphatidylinositol-4 kinase III alpha (PI4KIIIα), as being required for HCV replication. Consistent with elevated levels of the PI4KIIIα product phosphatidylinositol-4-phosphate (PI4P) detected in HCV-infected cultured hepatocytes and liver tissue from chronic hepatitis C patients, the enzymatic activity of PI4KIIIα was critical for HCV replication. Viral nonstructural protein 5A (NS5A) was found to interact with PI4KIIIα and stimulate its kinase activity. The absence of PI4KIIIα activity induced a dramatic change in the ultrastructural morphology of the membranous HCV replication complex. Our analysis suggests that the direct activation of a lipid kinase by HCV NS5A contributes critically to the integrity of the membranous viral replication complex.

  19. Enhanced phosphodiesteratic breakdown and turnover of phosphoinositides during reperfusion of ischemic rat heart.

    PubMed

    Otani, H; Prasad, M R; Engelman, R M; Otani, H; Cordis, G A; Das, D K

    1988-11-01

    In this study, we examined phosphoinositide metabolism during ischemia and reperfusion using an isolated and perfused rat heart. When myocardial phosphoinositides were prelabeled with [3H]inositol, reperfusion after 30 minutes of normothermic global ischemia resulted in significant accumulations of radiolabeled inositol phosphate, inositol bisphosphate, and inositol trisphosphate. Isotopic incorporation of [3H]inositol into phosphatidylinositol, phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-bisphosphate was increased significantly in the heart reperfused with [3H]inositol after 30 minutes of ischemia compared with that perfused with [3H]inositol after 30 minutes of nonischemic perfusion. However, isotopic incorporation of [3H]glycerol into diacylglycerol, phosphatidic acid, and all of the three phosphoinositides was diminished in the reperfused hearts. Reperfusion of the ischemic heart prelabeled with [14C]arachidonic acid resulted in significant increases in [14C]diacylglycerol and [14C]phosphatidic acid. The enhanced accumulations of [3H]inositol phosphates during reperfusion were not affected by treatment with prazosin plus atropine or indomethacin, but were inhibited by hypoxic reperfusion, reperfusion with Ca2+-free buffer, or by mepacrine. These results suggest that myocardial reperfusion stimulates phosphodiesteratic breakdown and turnover of phosphoinositides, and increased Ca2+ influx caused by reperfusion may be involved in the mechanism of stimulation of phosphatidylinositol-specific phospholipase C activity in the rat heart.

  20. Mutations in Escherichia coli aceE and ribB genes allow survival of strains defective in the first step of the isoprenoid biosynthesis pathway.

    PubMed

    Perez-Gil, Jordi; Uros, Eva Maria; Sauret-Güeto, Susanna; Lois, L Maria; Kirby, James; Nishimoto, Minobu; Baidoo, Edward E K; Keasling, Jay D; Boronat, Albert; Rodriguez-Concepcion, Manuel

    2012-01-01

    A functional 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is required for isoprenoid biosynthesis and hence survival in Escherichia coli and most other bacteria. In the first two steps of the pathway, MEP is produced from the central metabolic intermediates pyruvate and glyceraldehyde 3-phosphate via 1-deoxy-D-xylulose 5-phosphate (DXP) by the activity of the enzymes DXP synthase (DXS) and DXP reductoisomerase (DXR). Because the MEP pathway is absent from humans, it was proposed as a promising new target to develop new antibiotics. However, the lethal phenotype caused by the deletion of DXS or DXR was found to be suppressed with a relatively high efficiency by unidentified mutations. Here we report that several mutations in the unrelated genes aceE and ribB rescue growth of DXS-defective mutants because the encoded enzymes allowed the production of sufficient DXP in vivo. Together, this work unveils the diversity of mechanisms that can evolve in bacteria to circumvent a blockage of the first step of the MEP pathway.

  1. Hydrolyzed polyacrylamide grafted maize starch based microbeads: application in pH responsive drug delivery.

    PubMed

    Setty, C Mallikarjuna; Deshmukh, Anand S; Badiger, Aravind M

    2014-09-01

    The present study details the synthesis, characterization and pharmaceutical application of hydrolysed polyacrylamide grafted maize starch (HPam-g-MS) as promising polymeric material for the development of pH responsive microbeads. Different grades of graft copolymer were synthesized by changing the net microwave irradiation time, while keeping all other factors constant. Acute oral toxicity study performed in rodents ensured the bio-safety of graft copolymer for clinical application. Various batches of aceclofenac loaded microbeads were prepared by ionic gelation method using synthesized graft copolymers and evaluated for formulation parameters. FTIR spectroscopy confirmed the chemical compatibility between drug and graft copolymer. Results of in vitro release study (USP type-II) carried out in two different pH media (pH 1.2 acid buffer and pH 7.4 phosphate buffer) showed that release rate of drug from developed microbeads was a function of both: (a) surrounding pH and (b) the matrix composition. The drug release was relatively higher at alkaline pH as compared to acidic pH and this feature is desirable from viewpoint of site specific drug delivery. A direct correlation was observed between percentage grafting and microbeads performance and it presents a scope for further research on application and optimization of HPam-g-MS based microbeads as drug delivery carriers.

  2. Nucleocytoplasmic Shuttling of the Golgi Phosphatidylinositol 4-Kinase Pik1 Is Regulated by 14-3-3 Proteins and Coordinates Golgi Function with Cell Growth

    PubMed Central

    Demmel, Lars; Beck, Mike; Klose, Christian; Schlaitz, Anne-Lore; Gloor, Yvonne; Hsu, Peggy P.; Havlis, Jan; Shevchenko, Andrej; Krause, Eberhard; Kalaidzidis, Yannis

    2008-01-01

    The yeast phosphatidylinositol 4-kinase Pik1p is essential for proliferation, and it controls Golgi homeostasis and transport of newly synthesized proteins from this compartment. At the Golgi, phosphatidylinositol 4-phosphate recruits multiple cytosolic effectors involved in formation of post-Golgi transport vesicles. A second pool of catalytically active Pik1p localizes to the nucleus. The physiological significance and regulation of this dual localization of the lipid kinase remains unknown. Here, we show that Pik1p binds to the redundant 14-3-3 proteins Bmh1p and Bmh2p. We provide evidence that nucleocytoplasmic shuttling of Pik1p involves phosphorylation and that 14-3-3 proteins bind Pik1p in the cytoplasm. Nutrient deprivation results in relocation of Pik1p from the Golgi to the nucleus and increases the amount of Pik1p–14-3-3 complex, a process reversed upon restored nutrient supply. These data suggest a role of Pik1p nucleocytoplasmic shuttling in coordination of biosynthetic transport from the Golgi with nutrient signaling. PMID:18172025

  3. Type III phosphatidylinositol 4 kinases: structure, function, regulation, signalling and involvement in disease.

    PubMed

    Dornan, Gillian L; McPhail, Jacob A; Burke, John E

    2016-02-01

    Many important cellular functions are regulated by the selective recruitment of proteins to intracellular membranes mediated by specific interactions with lipid phosphoinositides. The enzymes that generate lipid phosphoinositides therefore must be properly positioned and regulated at their correct cellular locations. Phosphatidylinositol 4 kinases (PI4Ks) are key lipid signalling enzymes, and they generate the lipid species phosphatidylinositol 4-phosphate (PI4P), which plays important roles in regulating physiological processes including membrane trafficking, cytokinesis and organelle identity. PI4P also acts as the substrate for the generation of the signalling phosphoinositides phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3). PI4Ks also play critical roles in a number of pathological processes including mediating replication of a number of pathogenic RNA viruses, and in the development of the parasite responsible for malaria. Key to the regulation of PI4Ks is their regulation by a variety of both host and viral protein-binding partners. We review herein our current understanding of the structure, regulatory interactions and role in disease of the type III PI4Ks.

  4. Targeting Plasmodium PI(4)K to eliminate malaria.

    PubMed

    McNamara, Case W; Lee, Marcus C S; Lim, Chek Shik; Lim, Siau Hoi; Roland, Jason; Nagle, Advait; Simon, Oliver; Yeung, Bryan K S; Chatterjee, Arnab K; McCormack, Susan L; Manary, Micah J; Zeeman, Anne-Marie; Dechering, Koen J; Kumar, T R Santha; Henrich, Philipp P; Gagaring, Kerstin; Ibanez, Maureen; Kato, Nobutaka; Kuhen, Kelli L; Fischli, Christoph; Rottmann, Matthias; Plouffe, David M; Bursulaya, Badry; Meister, Stephan; Rameh, Lucia; Trappe, Joerg; Haasen, Dorothea; Timmerman, Martijn; Sauerwein, Robert W; Suwanarusk, Rossarin; Russell, Bruce; Renia, Laurent; Nosten, Francois; Tully, David C; Kocken, Clemens H M; Glynne, Richard J; Bodenreider, Christophe; Fidock, David A; Diagana, Thierry T; Winzeler, Elizabeth A

    2013-12-12

    Achieving the goal of malaria elimination will depend on targeting Plasmodium pathways essential across all life stages. Here we identify a lipid kinase, phosphatidylinositol-4-OH kinase (PI(4)K), as the target of imidazopyrazines, a new antimalarial compound class that inhibits the intracellular development of multiple Plasmodium species at each stage of infection in the vertebrate host. Imidazopyrazines demonstrate potent preventive, therapeutic, and transmission-blocking activity in rodent malaria models, are active against blood-stage field isolates of the major human pathogens P. falciparum and P. vivax, and inhibit liver-stage hypnozoites in the simian parasite P. cynomolgi. We show that imidazopyrazines exert their effect through inhibitory interaction with the ATP-binding pocket of PI(4)K, altering the intracellular distribution of phosphatidylinositol-4-phosphate. Collectively, our data define PI(4)K as a key Plasmodium vulnerability, opening up new avenues of target-based discovery to identify drugs with an ideal activity profile for the prevention, treatment and elimination of malaria.

  5. Type II PI4-kinases control Weibel-Palade body biogenesis and von Willebrand factor structure in human endothelial cells.

    PubMed

    Lopes da Silva, Mafalda; O'Connor, Marie N; Kriston-Vizi, Janos; White, Ian J; Al-Shawi, Raya; Simons, J Paul; Mössinger, Julia; Haucke, Volker; Cutler, Daniel F

    2016-05-15

    Weibel-Palade bodies (WPBs) are endothelial storage organelles that mediate the release of molecules involved in thrombosis, inflammation and angiogenesis, including the pro-thrombotic glycoprotein von Willebrand factor (VWF). Although many protein components required for WPB formation and function have been identified, the role of lipids is almost unknown. We examined two key phosphatidylinositol kinases that control phosphatidylinositol 4-phosphate levels at the trans-Golgi network, the site of WPB biogenesis. RNA interference of the type II phosphatidylinositol 4-kinases PI4KIIα and PI4KIIβ in primary human endothelial cells leads to formation of an increased proportion of short WPB with perturbed packing of VWF, as exemplified by increased exposure of antibody-binding sites. When stimulated with histamine, these cells release normal levels of VWF yet, under flow, form very few platelet-catching VWF strings. In PI4KIIα-deficient mice, immuno-microscopy revealed that VWF packaging is also perturbed and these mice exhibit increased blood loss after tail cut compared to controls. This is the first demonstration that lipid kinases can control the biosynthesis of VWF and the formation of WPBs that are capable of full haemostatic function.

  6. Multi-Substrate Terpene Synthases: Their Occurrence and Physiological Significance

    PubMed Central

    Pazouki, Leila; Niinemets, Ülo

    2016-01-01

    Terpene synthases are responsible for synthesis of a large number of terpenes in plants using substrates provided by two distinct metabolic pathways, the mevalonate-dependent pathway that is located in cytosol and has been suggested to be responsible for synthesis of sesquiterpenes (C15), and 2-C-methyl-D-erythritol-4-phosphate pathway located in plastids and suggested to be responsible for the synthesis of hemi- (C5), mono- (C10), and diterpenes (C20). Recent advances in characterization of genes and enzymes responsible for substrate and end product biosynthesis as well as efforts in metabolic engineering have demonstrated existence of a number of multi-substrate terpene synthases. This review summarizes the progress in the characterization of such multi-substrate terpene synthases and suggests that the presence of multi-substrate use might have been significantly underestimated. Multi-substrate use could lead to important changes in terpene product profiles upon substrate profile changes under perturbation of metabolism in stressed plants as well as under certain developmental stages. We therefore argue that multi-substrate use can be significant under physiological conditions and can result in complicate modifications in terpene profiles. PMID:27462341

  7. Involvement of Sac1 phosphoinositide phosphatase in the metabolism of phosphatidylserine in the yeast Saccharomyces cerevisiae.

    PubMed

    Tani, Motohiro; Kuge, Osamu

    2014-04-01

    Sac1 is a phosphoinositide phosphatase that preferentially dephosphorylates phosphatidylinositol 4-phosphate. Mutation of SAC1 causes not only the accumulation of phosphoinositides but also reduction of the phosphatidylserine (PS) level in the yeast Saccharomyces cerevisiae. In this study, we characterized the mechanism underlying the PS reduction in SAC1-deleted cells. Incorporation of (32) P into PS was significantly delayed in sac1∆ cells. Such a delay was also observed in SAC1- and PS decarboxylase gene-deleted cells, suggesting that the reduction in the PS level is caused by a reduction in the rate of biosynthesis of PS. A reduction in the PS level was also observed with repression of STT4 encoding phosphatidylinositol 4-kinase or deletion of VPS34 encoding phophatidylinositol 3-kinase. However, the combination of mutations of SAC1 and STT4 or VPS34 did not restore the reduced PS level, suggesting that both the synthesis and degradation of phosphoinositides are important for maintenance of the PS level. Finally, we observed an abnormal PS distribution in sac1∆ cells when a specific probe for PS was expressed. Collectively, these results suggested that Sac1 is involved in the maintenance of a normal rate of biosynthesis and distribution of PS.

  8. A PRELIMINARY STUDY OF THE REDUCING INTENSITY OF LUMINOUS BACTERIA

    PubMed Central

    Harvey, E. Newton

    1929-01-01

    The effect of a series of redox indicators and systems has been tested with a suspension of luminous bacteria (B. fischeri) in M/4 phosphate buffer of PH = 7.6. The indicators behave as expected from their position in the redox series, the most positive being reduced rapidly even in presence of air and before luminescence of the bacteria disappears, those of intermediate position at the time luminescence disappears, and the more negative only long after the luminescence had ceased, due to utilization of oxygen by the bacterial respiration. Indigo monosulphonate was the only indicator not reduced on long standing of a bacterial suspension. The aerobic redox potential may be placed at an RH = 18–20 and the anaerobic potential at an RH = 8–10. Ferricyanides do not affect luminescence and behave as if they could not penetrate the bacterial cell. Quinone and the napthoquinones cause progressive dimming of luminescence in any concentration which affects the light but it cannot be definitely stated that this is due to rapid oxidation of luciferin although it seems likely in the case of quinone. Some indophenols dim the luminescence at first, followed by return of brightness, which is interpreted to mean rapid oxidation of luciferin while the indophenol is unreduced, more luciferin production after reduction of indophenol. The more negative redox systems do not affect the luminescence. Investigation of indicator reduction and luminescence is being continued. PMID:19872505

  9. Fruit carotenoid-deficient mutants in tomato reveal a function of the plastidial isopentenyl diphosphate isomerase (IDI1) in carotenoid biosynthesis.

    PubMed

    Pankratov, Ilya; McQuinn, Ryan; Schwartz, Jochanan; Bar, Einat; Fei, Zhangjun; Lewinsohn, Efraim; Zamir, Dani; Giovannoni, James J; Hirschberg, Joseph

    2016-10-01

    Isoprenoids consist of a large class of compounds that are present in all living organisms. They are derived from the 5C building blocks isopentenyl diphosphate (IDP) and its isomer dimethylallyl diphosphate (DMADP). In plants, IDP is synthesized in the cytoplasm from mevalonic acid via the MVA pathway, and in plastids from 2-C-methyl-d-erythritol-4-phosphate through the MEP pathway. The enzyme IDP isomerase (IDI) catalyzes the interconversion between IDP and DMADP. Most plants contain two IDI enzymes, the functions of which are characteristically compartmentalized in the cells. Carotenoids are isoprenoids that play essential roles in photosynthesis and provide colors to flowers and fruits. They are synthesized in the plastids via the MEP pathway. Fruits of Solanum lycopersicum (tomato) accumulate high levels of the red carotene lycopene. We have identified mutations in tomato that reduce overall carotenoid accumulation in fruits. Four alleles of a locus named FRUIT CAROTENOID DEFICIENT 1 (fcd1) were characterized. Map-based cloning of fcd1 indicated that this gene encodes the plastidial enzyme IDI1. Lack of IDI1 reduced the concentration of carotenoids in fruits, flowers and cotyledons, but not in mature leaves. These results indicate that the plastidial IDI plays an important function in carotenoid biosynthesis, thus highlighting its role in optimizing the ratio between IDP and DMADP as precursors for different downstream isoprenoid pathways.

  10. Isopentenyl diphosphate and dimethylallyl diphosphate/isopentenyl diphosphate ratio measured with recombinant isopentenyl diphosphate isomerase and isoprene synthase.

    PubMed

    Zhou, Changfang; Li, Ziru; Wiberley-Bradford, Amy E; Weise, Sean E; Sharkey, Thomas D

    2013-09-15

    Isopentenyl diphosphate (IDP) and its isomer dimethylallyl diphosphate (DMADP) are building units for all isoprenoids; thus, intracellular pool sizes of IDP and DMADP play important roles in living organisms. Several methods have been used to quantify the amount of DMADP or the combined amount of IDP plus DMADP, but measuring the DMADP/IDP ratio has been difficult. In this study, a method was developed to measure the ratio of DMADP/IDP. Catalyzed by a recombinant IDP isomerase (IDI) together with a recombinant isoprene synthase (IspS), IDP was converted to isoprene, which was then detected by chemiluminescence. With this method, the in vitro equilibrium ratio of DMADP/IDP was found to be 2.11:1. IDP and DMADP pools were significantly increased in Escherichia coli transformed with methylerythritol 4-phosphate pathway genes; the ratio of DMADP/IDP was 3.85. An E. coli strain transformed with IspS but no additional IDI had a lower DMADP level and a DMADP/IDP ratio of 1.05. Approximately 90% of the IDP and DMADP pools in light-adapted kudzu leaves were light dependent and so presumably were located in the chloroplasts; the DMADP/IDP ratios in chloroplasts and cytosol were the same as the in vitro ratio (2.04 in the light and 2.32 in the dark).

  11. Non-enzymatic glycolysis and pentose phosphate pathway-like reactions in a plausible Archean ocean.

    PubMed

    Keller, Markus A; Turchyn, Alexandra V; Ralser, Markus

    2014-04-25

    The reaction sequences of central metabolism, glycolysis and the pentose phosphate pathway provide essential precursors for nucleic acids, amino acids and lipids. However, their evolutionary origins are not yet understood. Here, we provide evidence that their structure could have been fundamentally shaped by the general chemical environments in earth's earliest oceans. We reconstructed potential scenarios for oceans of the prebiotic Archean based on the composition of early sediments. We report that the resultant reaction milieu catalyses the interconversion of metabolites that in modern organisms constitute glycolysis and the pentose phosphate pathway. The 29 observed reactions include the formation and/or interconversion of glucose, pyruvate, the nucleic acid precursor ribose-5-phosphate and the amino acid precursor erythrose-4-phosphate, antedating reactions sequences similar to that used by the metabolic pathways. Moreover, the Archean ocean mimetic increased the stability of the phosphorylated intermediates and accelerated the rate of intermediate reactions and pyruvate production. The catalytic capacity of the reconstructed ocean milieu was attributable to its metal content. The reactions were particularly sensitive to ferrous iron Fe(II), which is understood to have had high concentrations in the Archean oceans. These observations reveal that reaction sequences that constitute central carbon metabolism could have been constrained by the iron-rich oceanic environment of the early Archean. The origin of metabolism could thus date back to the prebiotic world.

  12. Colors in the dark

    PubMed Central

    Rodríguez-Villalón, Antía; Gas, Elisabet

    2009-01-01

    Carotenoids are plastidial isoprenoid pigments essential for plant life. High carotenoid levels are found in chloroplasts and chromoplasts, but they are also produced in the etioplasts of seedlings that germinate in the dark. Our recent work has shown that an enhanced production of carotenoids in plastids of dark-grown Arabidopsis thaliana seedlings results in an improved transition to photosynthetic development (greening) upon illumination, illustrating the relevance of regulating etioplast carotenoid biosynthesis for plant fitness. We showed that the biosynthesis of etioplast carotenoids is controlled at the level of phytoene synthase (PSY), the enzyme catalyzing the first committed step of the pathway. Upregulation of PSY is necessary and sufficient to increase the production of carotenoids in dark-grown seedlings, in part because it triggers a feedback mechanism leading to the post-transcriptional accumulation of flux-controlling enzymes of the methylerythritol 4-phosphate (MEP) pathway, which synthesizes the substrates for PSY activity. Based on these and other recent data on the molecular mechanisms controlling deetiolation, we propose a model for the regulation of carotenoid biosynthesis in etioplasts. PMID:19826226

  13. Jasmonate-induced biosynthesis of andrographolide in Andrographis paniculata.

    PubMed

    Sharma, Shiv Narayan; Jha, Zenu; Sinha, Rakesh Kumar; Geda, Arvind Kumar

    2015-02-01

    Andrographolide is a prominent secondary metabolite found in Andrographis paniculata that exhibits enormous pharmacological effects. In spite of immense value, the normal biosynthesis of andrographolide results in low amount of the metabolite. To induce the biosynthesis of andrographolide, we attempted elicitor-induced activation of andrographolide biosynthesis in cell cultures of A. paniculata. This was carried out by using methyl jasmonate (MeJA) as an elicitor. Among the various concentrations of MeJA tested at different time periods, 5 µM MeJA yielded 5.25 times more andrographolide content after 24 h of treatment. The accumulation of andrographolide was correlated with the expression level of known regulatory genes (hmgs, hmgr, dxs, dxr, isph and ggps) of mevalonic acid (MVA) and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways. These results established the involvement of MeJA in andrographolide biosynthesis by inducing the transcription of its biosynthetic pathways genes. The coordination of isph, ggps and hmgs expression highly influenced the andrographolide biosynthesis.

  14. Development and validation of an equilibrium model for struvite formation with calcium co-precipitation

    NASA Astrophysics Data System (ADS)

    Lee, Sang-Hun; Yoo, Byeoung-Hak; Lim, Seung Joo; Kim, Tak-Hyun; Kim, Sun-Kyoung; Kim, Jun Young

    2013-06-01

    This study developed an equilibrium model to predict the P recovery and struvite amounts by newly incorporating two separate equilibrium constants on the struvite formation with HPO42- and PO43-, as well as free ammonium (NH4+), phosphate (PO4), magnesium (Mg2+), and calcium (Ca2+) ion species. The equilibrium struvite reaction and its solubility constant with HPO42- species was verified by deriving a reasonable correlation between solution pH and the conditional solubility products that were obtained from the equilibrium reaction. Also, based on the Visual MINTEQ software program, the potentially precipitated Ca phosphates and struvite precipitates were selected, and these compounds were utilized as target precipitants for the modeling to simulate P recovery and struvite formation under the competitive inhibition of Ca ions. The resultant simulated P recovery data were validated by experimental data with synthetic wastewater. The model data showed good agreement with the experimental results (R2>95%). The model also confirmed that the purity of struvite in the precipitate and the pH that maximizes the struvite fraction are dependent on the initial concentrations of NH4+, Mg2+, and PO4. Because only PO43-, not HPO42-, was regarded in Ca precipitation, Ca phosphate precipitation was underestimated as compared with the experimental results.

  15. Phosphoinositides in the hepatitis C virus life cycle.

    PubMed

    Bishé, Bryan; Syed, Gulam; Siddiqui, Aleem

    2012-10-19

    Eukaryotes possess seven different phosphoinositides (PIPs) that help form the unique signatures of various intracellular membranes. PIPs serve as docking sites for the recruitment of specific proteins to mediate membrane alterations and integrate various signaling cascades. The spatio-temporal regulation of PI kinases and phosphatases generates distinct intracellular hubs of PIP signaling. Hepatitis C virus (HCV), like other plus-strand RNA viruses, promotes the rearrangement of intracellular membranes to assemble viral replication complexes. HCV stimulates enrichment of phosphatidylinositol 4-phosphate (PI4P) pools near endoplasmic reticulum (ER) sites by activating PI4KIIIα, the kinase responsible for generation of ER-specific PI4P pools. Inhibition of PI4KIIIα abrogates HCV replication. PI4P, the most abundant phosphoinositide, predominantly localizes to the Golgi and plays central roles in Golgi secretory functions by recruiting effector proteins involved in transport vesicle generation. The PI4P effector proteins also include the lipid-transfer and structural proteins such as ceramide transfer protein (CERT), oxysterol binding protein (OSBP) and Golgi phosphoprotein 3 (GOLPH3) that help maintain Golgi-membrane composition and structure. Depletion of Golgi-specific PI4P pools by silencing PI4KIIIβ, expression of dominant negative CERT and OSBP mutants, or silencing GOLPH3 perturb HCV secretion. In this review we highlight the role of PIPs and specifically PI4P in the HCV life cycle.

  16. Antimicrobial mechanism of theaflavins: They target 1-deoxy-D-xylulose 5-phosphate reductoisomerase, the key enzyme of the MEP terpenoid biosynthetic pathway

    PubMed Central

    Hui, Xian; Yue, Qiao; Zhang, Dan-Dan; Li, Heng; Yang, Shao-Qing; Gao, Wen-Yun

    2016-01-01

    1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is the first committed enzyme in the 2-methyl-D-erythritol 4-phosphate (MEP) terpenoid biosynthetic pathway and is also a validated antimicrobial target. Theaflavins, which are polyphenolic compounds isolated from fermented tea, possess a wide range of pharmacological activities, especially an antibacterial effect, but little has been reported on their modes of antimicrobial action. To uncover the antibacterial mechanism of theaflavins and to seek new DXR inhibitors from natural sources, the DXR inhibitory activity of theaflavins were investigated in this study. The results show that all four theaflavin compounds could specifically suppress the activity of DXR, with theaflavin displaying the lowest effect against DXR (IC50 162.1 μM) and theaflavin-3,3′-digallate exhibiting the highest (IC50 14.9 μM). Moreover, determination of inhibition kinetics of the theaflavins demonstrates that they are non-competitive inhibitors of DXR against 1-deoxy-D-xylulose 5-phosphate (DXP) and un-competitive inhibitors with respect to NADPH. The possible interactions between DXR and the theaflavins were simulated via docking experiments. PMID:27941853

  17. The biochemical modification of the erythrocyte membranes from women with ovarian cancer.

    PubMed Central

    Kopczyński, Z.; Kuźniak, J.; Thielemann, A.; Kaczmarek, J.; Rybczyńska, M.

    1998-01-01

    The aim of our work was quantitative evaluation of the protein and phospholipid fractions of mature erythrocyte membranes separated from women with ovarian cancer. Blood was sampled from 30 women with ovarian cancer, aged 24-79 years, in the third stage of clinical progression of the disease. Phospholipids were separated from membranes by Müller's acidic extraction method and analysed in thin-layer two-dimensional chromatography. On the silica gel plates nine fractions of phospholipids were separated: sphingomyelin (SPH), phosphatidylethanolamine (PE), phosphatidlyserine (PS), phosphatidylcholine (PC), lysophosphatidylcholine (LPC), phosphatidic acid (PA), phosphatidylinositol (Ptd Ins), phosphatidylinositol-4-phosphate (Ptd Ins-4-P), phosphatidylinositol-4,5-diphosphate (Ptd Ins-4,5-P2). The activity of phospholipase C in erythrocyte membranes was determined by Akhrem's spectrophotometric method. Membrane proteins were separated by polyacrylamide gel electrophoresis, SDS-PAGE. It was shown that PS, SPH, LPC and PA fractions were significantly diminished. The concentration of Ptd Ins-4-P and Ptd Ins-4,5-P2 was significantly increased with simultaneous reduction in Ptd Ins level. The inhibition of phospholipase C reached 80%. The quantitative protein evaluation showed a statistically significant decrease in spectrin and a significant increase in 4.1 protein. The quantitative changes, observed in phospholipid and protein fractions, led to the restructuring of the erythrocyte membrane cytoskeleton, which may be connected to increased susceptibility to haemolysis of red blood cells. Images Figure 3 PMID:9716028

  18. Recruitment of arfaptins to the trans-Golgi network by PI(4)P and their involvement in cargo export

    PubMed Central

    Cruz-Garcia, David; Ortega-Bellido, Maria; Scarpa, Margherita; Villeneuve, Julien; Jovic, Marko; Porzner, Marc; Balla, Tamas; Seufferlein, Thomas; Malhotra, Vivek

    2013-01-01

    The BAR (Bin/Amphiphysin/Rvs) domain proteins arfaptin1 and arfaptin2 are localized to the trans-Golgi network (TGN) and, by virtue of their ability to sense and/or generate membrane curvature, could play an important role in the biogenesis of transport carriers. We report that arfaptins contain an amphipathic helix (AH) preceding the BAR domain, which is essential for their binding to phosphatidylinositol 4-phosphate (PI(4)P)-containing liposomes and the TGN of mammalian cells. The binding of arfaptin1, but not arfaptin2, to PI(4)P is regulated by protein kinase D (PKD) mediated phosphorylation at Ser100 within the AH. We also found that only arfaptin1 is required for the PKD-dependent trafficking of chromogranin A by the regulated secretory pathway. Altogether, these findings reveal the importance of PI(4)P and PKD in the recruitment of arfaptins at the TGN and their requirement in the events leading to the biogenesis of secretory storage granules. PMID:23695357

  19. Engineering of a Synthetic Metabolic Pathway for the Assimilation of (d)-Xylose into Value-Added Chemicals.

    PubMed

    Cam, Yvan; Alkim, Ceren; Trichez, Debora; Trebosc, Vincent; Vax, Amélie; Bartolo, François; Besse, Philippe; François, Jean Marie; Walther, Thomas

    2016-07-15

    A synthetic pathway for (d)-xylose assimilation was stoichiometrically evaluated and implemented in Escherichia coli strains. The pathway proceeds via isomerization of (d)-xylose to (d)-xylulose, phosphorylation of (d)-xylulose to obtain (d)-xylulose-1-phosphate (X1P), and aldolytic cleavage of the latter to yield glycolaldehyde and DHAP. Stoichiometric analyses showed that this pathway provides access to ethylene glycol with a theoretical molar yield of 1. Alternatively, both glycolaldehyde and DHAP can be converted to glycolic acid with a theoretical yield that is 20% higher than for the exclusive production of this acid via the glyoxylate shunt. Simultaneous expression of xylulose-1 kinase and X1P aldolase activities, provided by human ketohexokinase-C and human aldolase-B, respectively, restored growth of a (d)-xylulose-5-kinase mutant on xylose. This strain produced ethylene glycol as the major metabolic endproduct. Metabolic engineering provided strains that assimilated the entire C2 fraction into the central metabolism or that produced 4.3 g/L glycolic acid at a molar yield of 0.9 in shake flasks.

  20. Mechanism of RNA 2',3'-cyclic phosphate end healing by T4 polynucleotide kinase-phosphatase.

    PubMed

    Das, Ushati; Shuman, Stewart

    2013-01-07

    T4 polynucleotide kinase-phosphatase (Pnkp) exemplifies a family of enzymes with 5'-kinase and 3'-phosphatase activities that function in nucleic acid repair. The polynucleotide 3'-phosphatase reaction is executed by the Pnkp C-terminal domain, which belongs to the DxDxT acylphosphatase superfamily. The 3'-phosphatase reaction entails formation and hydrolysis of a covalent enzyme-(Asp165)-phosphate intermediate, driven by general acid-base catalyst Asp167. We report that Pnkp also has RNA 2'-phosphatase activity that requires Asp165 and Asp167. The physiological substrate for Pnkp phosphatase is an RNA 2',3'-cyclic phosphate end (RNA > p), but the pathway of cyclic phosphate removal and its enzymic requirements are undefined. Here we find that Pnkp reactivity with RNA > p requires Asp165, but not Asp167. Whereas wild-type Pnkp transforms RNA > p to RNA(OH), mutant D167N converts RNA > p to RNA 3'-phosphate, which it sequesters in the phosphatase active site. In support of the intermediacy of an RNA phosphomonoester, the reaction of mutant S211A with RNA > p results in transient accumulation of RNAp en route to RNA(OH). Our results suggest that healing of 2',3'-cyclic phosphate ends is a four-step processive reaction: RNA > p + Pnkp → RNA-(3'-phosphoaspartyl)-Pnkp → RNA(3')p + Pnkp → RNA(OH) + phosphoaspartyl-Pnkp → P(i) + Pnkp.

  1. Structure and mechanism of the 2',3' phosphatase component of the bacterial Pnkp-Hen1 RNA repair system.

    PubMed

    Wang, Li Kai; Smith, Paul; Shuman, Stewart

    2013-06-01

    Pnkp is the end-healing and end-sealing component of an RNA repair system present in diverse bacteria from many phyla. Pnkp is composed of three catalytic modules: an N-terminal polynucleotide 5' kinase, a central 2',3' phosphatase and a C-terminal ligase. The phosphatase module is a Mn(2+)-dependent phosphodiesterase-monoesterase that dephosphorylates 2',3'-cyclic phosphate RNA ends. Here we report the crystal structure of the phosphatase domain of Clostridium thermocellum Pnkp with Mn(2+) and citrate in the active site. The protein consists of a core binuclear metallo-phosphoesterase fold (exemplified by bacteriophage λ phosphatase) embellished by distinctive secondary structure elements. The active site contains a single Mn(2+) in an octahedral coordination complex with Asp187, His189, Asp233, two citrate oxygens and a water. The citrate fills the binding site for the scissile phosphate, wherein it is coordinated by Arg237, Asn263 and His264. The citrate invades the site normally occupied by a second metal (engaged by Asp233, Asn263, His323 and His376), and thereby dislocates His376. A continuous tract of positive surface potential flanking the active site suggests an RNA binding site. The structure illuminates a large body of mutational data regarding the metal and substrate specificity of Clostridium thermocellum Pnkp phosphatase.

  2. Host PI(3,5)P2 activity is required for Plasmodium berghei growth during liver stage infection

    PubMed Central

    Thieleke-Matos, Carolina; da Silva, Mafalda Lopes; Cabrita-Santos, Laura; Pires, Cristiana F.; Ramalho, José S.; Ikonomov, Ognian; Seixas, Elsa; Shisheva, Assia; Seabra, Miguel C.; Barral, Duarte C.

    2014-01-01

    Malaria parasites go through an obligatory liver stage before they infect erythrocytes and cause disease symptoms. In the host hepatocytes, the parasite is enclosed by a parasitophorous vacuole membrane (PVM). Here, we dissected the interaction between the Plasmodium parasite and the host cell late endocytic pathway and show that parasite growth is dependent on the phosphoinositide 5-kinase (PIKfyve), which converts phosphatidylinositol 3-phosphate [PI(3)P] into phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] in the endosomal system. We found that inhibition of PIKfyve either by pharmacological or non-pharmacological means causes a delay in parasite growth. Moreover, we show that the PI(3,5)P2 effector protein TRPML1 that is involved in late endocytic membrane fusion, is present in vesicles closely contacting the PVM and is necessary for parasite growth. Thus, our studies suggest that the parasite PVM is able to fuse with host late endocytic vesicles in a PI(3,5)P2-dependent manner, allowing the exchange of material between the host and the parasite, which is essential for successful infection. PMID:24992508

  3. A Novel Mechanism of Ion Homeostasis and Salt Tolerance in Yeast: the Hal4 and Hal5 Protein Kinases Modulate the Trk1-Trk2 Potassium Transporter

    PubMed Central

    Mulet, Jose M.; Leube, Martin P.; Kron, Stephen J.; Rios, Gabino; Fink, Gerald R.; Serrano, Ramon

    1999-01-01

    The regulation of intracellular ion concentrations is a fundamental property of living cells. Although many ion transporters have been identified, the systems that modulate their activity remain largely unknown. We have characterized two partially redundant genes from Saccharomyces cerevisiae, HAL4/SAT4 and HAL5, that encode homologous protein kinases implicated in the regulation of cation uptake. Overexpression of these genes increases the tolerance of yeast cells to sodium and lithium, whereas gene disruptions result in greater cation sensitivity. These phenotypic effects of the mutations correlate with changes in cation uptake and are dependent on a functional Trk1-Trk2 potassium transport system. In addition, hal4 hal5 and trk1 trk2 mutants exhibit similar phenotypes: (i) they are deficient in potassium uptake; (ii) their growth is sensitive to a variety of toxic cations, including lithium, sodium, calcium, tetramethylammonium, hygromycin B, and low pH; and (iii) they exhibit increased uptake of methylammonium, an indicator of membrane potential. These results suggest that the Hal4 and Hal5 protein kinases activate the Trk1-Trk2 potassium transporter, increasing the influx of potassium and decreasing the membrane potential. The resulting loss in electrical driving force reduces the uptake of toxic cations and improves salt tolerance. Our data support a role for regulation of membrane potential in adaptation to salt stress that is mediated by the Hal4 and Hal5 kinases. PMID:10207057

  4. α-fur, an antisense RNA gene to fur in the extreme acidophile Acidithiobacillus ferrooxidans.

    PubMed

    Lefimil, C; Jedlicki, E; Holmes, D S

    2014-03-01

    A large non-coding RNA, termed α-Fur, of ~1000 nt has been detected in the extreme acidophile Acidithiobacillus ferrooxidans encoded on the antisense strand to the iron-responsive master regulator fur (ferric uptake regulator) gene. A promoter for α-fur was predicted bioinformatically and validated using gene fusion experiments. The promoter is situated within the coding region and in the same sense as proB, potentially encoding a glutamate 5-kinase. The 3' termination site of the α-fur transcript was determined by 3' rapid amplification of cDNA ends to lie 7 nt downstream of the start of transcription of fur. Thus, α-fur is antisense to the complete coding region of fur, including its predicted ribosome-binding site. The genetic context of α-fur is conserved in several members of the genus Acidithiobacillus but not in all acidophiles, indicating that it is monophyletic but not niche specific. It is hypothesized that α-Fur regulates the cellular level of Fur. This is the fourth example of an antisense RNA to fur, although it is the first in an extreme acidophile, and underscores the growing importance of cis-encoded non-coding RNAs as potential regulators involved in the microbial iron-responsive stimulon.

  5. Regulation of ion channels and transporters by AMP-activated kinase (AMPK)

    PubMed Central

    Lang, Florian; Föller, Michael

    2014-01-01

    The energy-sensing AMP-activated kinase AMPK ensures survival of energy-depleted cells by stimulating ATP production and limiting ATP utilization. Both energy production and energy consumption are profoundly influenced by transport processes across the cell membane including channels, carriers and pumps. Accordingly, AMPK is a powerful regulator of transport across the cell membrane. AMPK regulates diverse K+ channels, Na+ channels, Ca2+ release activated Ca2+ channels, Cl- channels, gap junctional channels, glucose carriers, Na+/H+-exchanger, monocarboxylate-, phosphate-, creatine-, amino acid-, peptide- and osmolyte-transporters, Na+/Ca2+-exchanger, H+-ATPase and Na+/K+-ATPase. AMPK activates ubiquitin ligase Nedd4–2, which labels several plasma membrane proteins for degradation. AMPK further regulates transport proteins by inhibition of Rab GTPase activating protein (GAP) TBC1D1. It stimulates phosphatidylinositol 3-phosphate 5-kinase PIKfyve and inhibits phosphatase and tensin homolog (PTEN) via glycogen synthase kinase 3β (GSK3β). Moreover, it stabilizes F-actin as well as downregulates transcription factor NF-κB. All those cellular effects serve to regulate transport proteins. PMID:24366036

  6. Rapid structural changes and acidification of guard cell vacuoles during stomatal closure require phosphatidylinositol 3,5-bisphosphate.

    PubMed

    Bak, Gwangbae; Lee, Eun-Jung; Lee, Yuree; Kato, Mariko; Segami, Shoji; Sze, Heven; Maeshima, Masayoshi; Hwang, Jae-Ung; Lee, Youngsook

    2013-06-01

    Rapid stomatal closure is essential for water conservation in plants and is thus critical for survival under water deficiency. To close stomata rapidly, guard cells reduce their volume by converting a large central vacuole into a highly convoluted structure. However, the molecular mechanisms underlying this change are poorly understood. In this study, we used pH-indicator dyes to demonstrate that vacuolar convolution is accompanied by acidification of the vacuole in fava bean (Vicia faba) guard cells during abscisic acid (ABA)-induced stomatal closure. Vacuolar acidification is necessary for the rapid stomatal closure induced by ABA, since a double mutant of the vacuolar H(+)-ATPase vha-a2 vha-a3 and vacuolar H(+)-PPase mutant vhp1 showed delayed stomatal closure. Furthermore, we provide evidence for the critical role of phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2] in changes in pH and morphology of the vacuole. Single and double Arabidopsis thaliana null mutants of phosphatidylinositol 3-phosphate 5-kinases (PI3P5Ks) exhibited slow stomatal closure upon ABA treatment compared with the wild type. Moreover, an inhibitor of PI3P5K reduced vacuolar acidification and convolution and delayed stomatal closure in response to ABA. Taken together, these results suggest that rapid ABA-induced stomatal closure requires PtdIns(3,5)P2, which is essential for vacuolar acidification and convolution.

  7. PGE2 induces angiogenesis via MT1-MMP-mediated activation of the TGFbeta/Alk5 signaling pathway.

    PubMed

    Alfranca, Arántzazu; López-Oliva, Juan Manuel; Genís, Laura; López-Maderuelo, Dolores; Mirones, Isabel; Salvado, Dolores; Quesada, Antonio J; Arroyo, Alicia G; Redondo, Juan Miguel

    2008-08-15

    The development of a new vascular network is essential for the onset and progression of many pathophysiologic processes. Cyclooxygenase-2 displays a proangiogenic activity in in vitro and in vivo models, mediated principally through its metabolite prostaglandin E(2) (PGE(2)). Here, we provide evidence for a novel signaling route through which PGE(2) activates the Alk5-Smad3 pathway in endothelial cells. PGE(2) induces Alk5-dependent Smad3 nuclear translocation and DNA binding, and the activation of this pathway involves the release of active TGFbeta from its latent form through a process mediated by the metalloproteinase MT1-MMP, whose membrane clustering is promoted by PGE(2). MT1-MMP-dependent transforming growth factor beta (TGFbeta) signaling through Alk5 is also required for PGE(2)-induced endothelial cord formation in vitro, and Alk5 kinase activity is required for PGE(2)-induced neovascularization in vivo. These findings identify a novel signaling pathway linking PGE(2) and TGFbeta, 2 effectors involved in tumor growth and angiogenesis, and reveal potential targets for the treatment of angiogenesis-related disorders.

  8. S-nitrosylation of cyclin-dependent kinase 5 (cdk5) regulates its kinase activity and dendrite growth during neuronal development.

    PubMed

    Zhang, Peng; Yu, Pei-Chun; Tsang, Anthony H K; Chen, Yu; Fu, Amy K Y; Fu, Wing-Yu; Chung, Kenny K; Ip, Nancy Y

    2010-10-27

    Precise regulation of cyclin-dependent kinase 5 (Cdk5), a member of the cyclin-dependent kinase family, is critical for proper neuronal development and functions. Cdk5 is activated through its association with the neuron-specific activator p35 or p39. Nonetheless, how its kinase activity is regulated in neurons is not well understood. In this study, we found that Cdk5 activity is regulated by S-nitrosylation, a post-translational modification of protein that affects a plethora of neuronal functions. S-nitrosylation of Cdk5 occurs at Cys83, which is one of the critical amino acids within the ATP-binding pocket of the kinase. Upon S-nitrosylation, Cdk5 exhibits reduced kinase activity, whereas mutation of Cys83 to Ala on Cdk5 renders the kinase refractory to such inhibition. Importantly, S-nitrosylated Cdk5 can be detected in the mouse brain, and blocking the S-nitrosylation of Cdk5 in cultured hippocampal neurons enhances dendritic growth and branching. Together, our findings reveal an important role of S-nitrosylation in regulating Cdk5 kinase activity and dendrite growth in neurons during development.

  9. Centrosome-Dependent Bypass of the DNA Damage Checkpoint by the Polo Kinase Cdc5.

    PubMed

    Ratsima, Hery; Serrano, Diego; Pascariu, Mirela; D'Amours, Damien

    2016-02-16

    Cell-cycle checkpoints are essential feedback mechanisms that promote genome integrity. However, in the face of unrepairable DNA lesions, bypass mechanisms can suppress checkpoint activity and allow cells to resume proliferation. The molecular mechanisms underlying this biological response are currently not understood. Taking advantage of unique separation-of-function mutants, we show that the Polo-like kinase (PLK) Cdc5 uses a phosphopriming-based interaction mechanism to suppress G2/M checkpoint arrest by targeting Polo kinase activity to centrosomes. We also show that key subunits of the evolutionarily conserved RSC complex are critical downstream effectors of Cdc5 activity in checkpoint suppression. Importantly, the lethality and checkpoint defects associated with loss of Cdc5 Polo box activity can be fully rescued by artificially anchoring Cdc5 kinase domain to yeast centrosomes. Collectively, our results highlight a previously unappreciated role for centrosomes as key signaling centers for the suppression of cell-cycle arrest induced by persistent or unrepairable DNA damage.

  10. Protein interaction module-assisted function X (PIMAX) approach to producing challenging proteins including hyperphosphorylated tau and active CDK5/p25 kinase complex.

    PubMed

    Sui, Dexin; Xu, Xinjing; Ye, Xuemei; Liu, Mengyu; Mianecki, Maxwell; Rattanasinchai, Chotirat; Buehl, Christopher; Deng, Xiexiong; Kuo, Min-Hao

    2015-01-01

    Many biomedically critical proteins are underrepresented in proteomics and biochemical studies because of the difficulty of their production in Escherichia coli. These proteins might possess posttranslational modifications vital to their functions, tend to misfold and be partitioned into bacterial inclusion bodies, or act only in a stoichiometric dimeric complex. Successful production of these proteins requires efficient interaction between these proteins and a specific "facilitator," such as a protein-modifying enzyme, a molecular chaperone, or a natural physical partner within the dimeric complex. Here we report the design and application of a protein interaction module-assisted function X (PIMAX) system that effectively overcomes these hurdles. By fusing two proteins of interest to a pair of well-studied protein-protein interaction modules, we were able to potentiate the association of these two proteins, resulting in successful production of an enzymatically active cyclin-dependent kinase complex and hyperphosphorylated tau protein, which is intimately linked to Alzheimer disease. Furthermore, using tau isoforms quantitatively phosphorylated by GSK-3β and CDK5 kinases via PIMAX, we demonstrated the hyperphosphorylation-stimulated tau oligomerization in vitro, paving the way for new Alzheimer disease drug discoveries. Vectors for PIMAX can be easily modified to meet the needs of different applications. This approach thus provides a convenient and modular suite with broad implications for proteomics and biomedical research.

  11. Robust Replication Control Is Generated by Temporal Gaps between Licensing and Firing Phases and Depends on Degradation of Firing Factor Sld2.

    PubMed

    Reusswig, Karl-Uwe; Zimmermann, Fabian; Galanti, Lorenzo; Pfander, Boris

    2016-10-04

    Temporal separation of DNA replication initiation into licensing and firing phases ensures the precise duplication of the genome during each cell cycle. Cyclin-dependent kinase (CDK) is known to generate this separation by activating firing factors and at the same time inhibiting licensing factors but may not be sufficient to ensure robust separation at transitions between both phases. Here, we show that a temporal gap separates the inactivation of firing factors from the re-activation of licensing factors during mitosis in budding yeast. We find that gap size critically depends on phosphorylation-dependent degradation of the firing factor Sld2 mediated by CDK, DDK, Mck1, and Cdc5 kinases and the ubiquitin-ligases Dma1/2. Stable mutants of Sld2 minimize the gap and cause increased genome instability in an origin-dependent manner when combined with deregulation of other replication regulators or checkpoint mechanisms. Robust separation of licensing and firing phases therefore appears indispensable to safeguard genome stability.

  12. Chemical Synthesis and Molecular Recognition of Phosphatase-Resistant Analogues of Phosphatidylinositol-3-phosphate

    PubMed Central

    Xu, Yong; Lee, Stephanie A.; Kutateladze, Tatiana G.; Sbrissa, Diego; Shisheva, Assia; Prestwich, Glenn D.

    2008-01-01

    The remodeling of phosphatidylinositol polyphosphates in cellular membranes by phosphatases and kinases orchestrates the signaling by these lipids in space and time. In order to provide chemical tools to study of the changes in cell physiology mediated by these lipids, three new metabolically-stabilized (ms) analogues of phosphatidylinositol-3-phosphate (PtdIns(3)P were synthesized. We describe herein the total asymmetric synthesis of 3-methylphosphonate, 3-monofluoromethylphosphonate and 3-phosphorothioate analogues of PtdIns(3)P. From differentially protected D-myo-inositol key intermediates, a versatile phosphoramidite reagent was employed in the synthesis of PtdIns(3)P analogues with diacylglyceryl moieties containing dioleoyl, dipalmitoyl and dibutyryl chains. In addition, we introduce a new phosphorlyation reagent, monofluoromethylphosphonyl chloride, which has general applications for the preparation of “pKa-matched” monofluorophosphonates. These ms-PtdIns(3)P analogues exhibited reduced binding activities with 15N-labelled FYVE and PX domains, as significant 1H and 15N chemical shift changes in the FYVE domain were induced by titrating ms-PtdIns(3)Ps into membrane-mimetic dodecylphosphocholine (DPC) micelles. In addition, the PtdIns(3)P analogues with dioleyl and dipalmitoyl chains were substrates for the 5-kinase enzyme PIKfyve; the corresponding phosphorylated ms-PI(3,5)P2 products were detected by radio-TLC analysis. PMID:16417379

  13. The insect peptide CopA3 inhibits lipopolysaccharide-induced macrophage activation.

    PubMed

    Nam, Hyo Jung; Oh, Ah Reum; Nam, Seung Taek; Kang, Jin Ku; Chang, Jong Soo; Kim, Dae Hong; Lee, Ji Hye; Hwang, Jae Sam; Shong, Ko Eun; Park, Mi Jung; Seok, Heon; Kim, Ho

    2012-10-01

    We recently demonstrated that the insect peptide CopA3 (LLCIALRKK), a disulfide-linked dimeric peptide, exerts antimicrobial and anti-inflammatory activities in a mouse colitis model. Here, we examined whether CopA3 inhibited activation of macrophages by LPS. Exposure of an unseparated mouse peritoneal cell population or isolated peritoneal macrophages to LPS markedly increased secretion of IL-6 and TNF-α; these effects were significantly inhibited by CopA3 treatment. The inhibitory effect of CopA3 was also evident in murine macrophage cell line, RAW 264.7. Western blotting revealed that LPS-induced activation of STAT1 and STAT5 in macrophages was significantly inhibited by CopA3. Inhibition of JAK (STAT1/STAT5 kinase) with AG490 markedly reduced the production of IL-6 and TNF-α in macrophages. Collectively, these observations suggest that CopA3 inhibits macrophage activation by inhibiting activating phosphorylations of the transcription factors, STAT1 and STAT5, and blocking subsequent production of IL-6 and TNF-α and indicate that CopA3 may be useful as an immune-modulating agent.

  14. Mus81-Mms4 Functions as a Single Heterodimer To Cleave Nicked Intermediates in Recombinational DNA Repair

    PubMed Central

    Schwartz, Erin K.; Wright, William D.; Ehmsen, Kirk T.; Evans, James E.; Stahlberg, Henning

    2012-01-01

    The formation of crossovers is a fundamental genetic process. The XPF-family endonuclease Mus81-Mms4 (Eme1) contributes significantly to crossing over in eukaryotes. A key question is whether Mus81-Mms4 can process Holliday junctions that contain four uninterrupted strands. Holliday junction cleavage requires the coordination of two active sites, necessitating the assembly of two Mus81-Mms4 heterodimers. Contrary to this expectation, we show that Saccharomyces cerevisiae Mus81-Mms4 exists as a single heterodimer both in solution and when bound to DNA substrates in vitro. Consistently, immunoprecipitation experiments demonstrate that Mus81-Mms4 does not multimerize in vivo. Moreover, chromatin-bound Mus81-Mms4 does not detectably form higher-order multimers. We show that Cdc5 kinase activates Mus81-Mms4 nuclease activity on 3′ flaps and Holliday junctions in vitro but that activation does not induce a preference for Holliday junctions and does not induce multimerization of the Mus81-Mms4 heterodimer. These data support a model in which Mus81-Mms4 cleaves nicked recombination intermediates such as displacement loops (D-loops), nicked Holliday junctions, or 3′ flaps but not intact Holliday junctions with four uninterrupted strands. We infer that Mus81-dependent crossing over occurs in a noncanonical manner that does not involve the coordinated cleavage of classic Holliday junctions. PMID:22645308

  15. Transient receptor potential melastatin 3 is a phosphoinositide-dependent ion channel.

    PubMed

    Badheka, Doreen; Borbiro, Istvan; Rohacs, Tibor

    2015-07-01

    Phosphoinositides are emerging as general regulators of the functionally diverse transient receptor potential (TRP) ion channel family. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) has been reported to positively regulate many TRP channels, but in several cases phosphoinositide regulation is controversial. TRP melastatin 3 (TRPM3) is a heat-activated ion channel that is also stimulated by chemical agonists, such as pregnenolone sulfate. Here, we used a wide array of approaches to determine the effects of phosphoinositides on TRPM3. We found that channel activity in excised inside-out patches decreased over time (rundown), an attribute of PI(4,5)P2-dependent ion channels. Channel activity could be restored by application of either synthetic dioctanoyl (diC8) or natural arachidonyl stearyl (AASt) PI(4,5)P2. The PI(4,5)P2 precursor phosphatidylinositol 4-phosphate (PI(4)P) was less effective at restoring channel activity. TRPM3 currents were also restored by MgATP, an effect which was inhibited by two different phosphatidylinositol 4-kinase inhibitors, or by pretreatment with a phosphatidylinositol-specific phospholipase C (PI-PLC) enzyme, indicating that MgATP acted by generating phosphoinositides. In intact cells, reduction of PI(4,5)P2 levels by chemically inducible phosphoinositide phosphatases or a voltage-sensitive 5'-phosphatase inhibited channel activity. Activation of PLC via muscarinic receptors also inhibited TRPM3 channel activity. Overall, our data indicate that TRPM3 is a phosphoinositide-dependent ion channel and that decreasing PI(4,5)P2 abundance limits its activity. As all other members of the TRPM family have also been shown to require PI(4,5)P2 for activity, our data establish PI(4,5)P2 as a general positive cofactor of this ion channel subfamily.

  16. Effect of long-term organic removal on ion exchange properties and performance during sewage tertiary treatment by conventional anion exchange resins.

    PubMed

    Sun, Jian; Li, Xiaofeng; Quan, Ying; Yin, Yunjun; Zheng, Shaokui

    2015-10-01

    This study evaluated the long-term dissolved organic matter (DOM), phosphorus and nitrogen removal performance of a commercially available conventional anion exchange resin (AER) from actual secondary effluent (SE) in a sewage treatment plant based on a pilot-scale operation (2.2 m(3) d(-1), 185 cycles, 37,000 bed volume, 1.5 years). Particular emphasis was given to the potential effect of DOM fouling on the ion exchange properties and performance during the long-term operation. Despite the large range of COD (15.6-33.5 mg L(-1)), BOD5 (3.0-5.6 mg L(-1)), DOC (6.5-24.2 mg L(-1)), and UV254 (UV absorption at 254 nm) (0.108-0.229 cm(-1)) levels in the SE, the removal efficiencies of the AER for the aforementioned parameters were 43±12%, 46±15%, 45±9%, and 72±4%, respectively. Based on three-dimensional fluorescence excitation-emission matrix data, i.e., the fluorescence intensities of four regions (peaks A-D), all organic components of the SE were effectively removed (peak A 74%, peak B 48%, peak C 55%, and peak D 45%) following the adsorption. The AER effluent still has considerable polycyclic aromatic hydrocarbons' ecological hazard on freshwater fishes when they were significantly removed from SE. The obvious DOM fouling on the AER, identified by color change, had no significant influence on the long-term removal of the representative inorganic anions (averaging 95±4% phosphate, 100±0% SO4(2-), and 62±17% NO3(-)) and AER properties (including total exchange capacity, moisture content, and true density). The conventional AER can produce high quality reclaimed water from SE at a low operational cost.

  17. A poly(l-glutamic acid)-combretastatin A4 conjugate for solid tumor therapy: Markedly improved therapeutic efficiency through its low tissue penetration in solid tumor.

    PubMed

    Liu, Tianzhou; Zhang, Dawei; Song, Wantong; Tang, Zhaohui; Zhu, Jiaming; Ma, Zhiming; Wang, Xudong; Chen, Xuesi; Tong, Ti

    2017-02-03

    Combretastatin A4 (CA4) is a leading agent in vascular disrupting strategies for tumor therapy. Although many small-molecule prodrugs of CA4 have been developed to improve its solubility, the overall therapeutic efficiency is moderate. A key reason for this is the reversible effect that CA4 has on tubulin as well as its rapid clearance from plasma and tissues. In this study, we proposed a poly(l-glutamic acid)-CA4 conjugate (PLG-CA4) nanomedicine to fulfill the requirements for fully liberating the potential of CA4 on tumor therapy. Enhanced accumulation and retention of CA4 in tumor tissue, especially, high distribution and gradual release around tumor blood vessels resulted in prolonged vascular disruption and markedly enhanced therapeutic efficiency. We examined and compared the therapeutic effect of PLG-CA4 and commercial combretastatin-A4 phosphate (CA4P) in a murine colon C26 tumor. PLG-CA4 showed significantly prolonged retention in plasma and tumor tissue. Most importantly, the PLG-CA4 was mainly distributed around the tumor vessels because of its low tissue penetration in solid tumor. Pathology tests showed that PLG-CA4 treatment resulted in persistent vascular disruption and tumor damage 72h after a single injection, this in contrast to CA4P treatment, which showed quick relapse at an equal dose. Tumor suppression tests showed that PLG-CA4 treatment resulted in a tumor suppression rate of 74%, which indicates a significant advantage when compared to tumor suppression rate of the CA4P group, which was 24%. This is the first time that an advantage of the polymeric CA4 nanomedicine with low tissue penetration for solid tumor therapy has been shown. Thus, the results presented in this study provide a new idea for enhancing the tumor therapeutic effect of vascular disrupting agents.

  18. Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

    PubMed Central

    Luchsinger, Charlotte; Rivera-Dictter, Andrés; Arriagada, Cecilia; Acuña, Diego; Aguilar, Marcelo; Cavieres, Viviana; Burgos, Patricia V.; Ehrenfeld, Pamela; Mardones, Gonzalo A.

    2016-01-01

    Golgi phosphoprotein 3 (GOLPH3) has been implicated in the development of carcinomas in many human tissues, and is currently considered a bona fide oncoprotein. Importantly, several tumor types show overexpression of GOLPH3, which is associated with tumor progress and poor prognosis. However, the underlying molecular mechanisms that connect GOLPH3 function with tumorigenicity are poorly understood. Experimental evidence shows that depletion of GOLPH3 abolishes transformation and proliferation of tumor cells in GOLPH3-overexpressing cell lines. Conversely, GOLPH3 overexpression drives transformation of primary cell lines and enhances mouse xenograft tumor growth in vivo. This evidence suggests that overexpression of GOLPH3 could result in distinct features of GOLPH3 in tumor cells compared to that of non-tumorigenic cells. GOLPH3 is a peripheral membrane protein mostly localized at the trans-Golgi network, and its association with Golgi membranes depends on binding to phosphatidylinositol-4-phosphate. GOLPH3 is also contained in a large cytosolic pool that rapidly exchanges with Golgi-associated pools. GOLPH3 has also been observed associated with vesicles and tubules arising from the Golgi, as well as other cellular compartments, and hence it has been implicated in several membrane trafficking events. Whether these and other features are typical to all different types of cells is unknown. Moreover, it remains undetermined how GOLPH3 acts as an oncoprotein at the Golgi. Therefore, to better understand the roles of GOLPH3 in cancer cells, we sought to compare some of its biochemical and cellular properties in the human breast cancer cell lines MCF7 and MDA-MB-231 with that of the non-tumorigenic breast human cell line MCF 10A. We found unexpected differences that support the notion that in different cancer cells, overexpression of GOLPH3 functions in diverse fashions, which may influence specific tumorigenic phenotypes. PMID:27123979

  19. LuxR- and acyl-homoserine-lactone-controlled non-lux genes define a quorum-sensing regulon in Vibrio fischeri.

    PubMed

    Callahan, S M; Dunlap, P V

    2000-05-01

    The luminescence (lux) operon (luxICDABEG) of the symbiotic bacterium Vibrio fischeri is regulated by the transcriptional activator LuxR and two acyl-homoserine lactone (acyl-HSL) autoinducers (the luxI-dependent 3-oxo-hexanoyl-HSL [3-oxo-C6-HSL] and the ainS-dependent octanoyl-HSL [C8-HSL]) in a population density-responsive manner called quorum sensing. To identify quorum-sensing-regulated (QSR) proteins different from those encoded by lux genes, we examined the protein patterns of V. fischeri quorum-sensing mutants defective in luxI, ainS, and luxR by two-dimensional polyacrylamide gel electrophoresis. Five non-Lux QSR proteins, QsrP, RibB, AcfA, QsrV, and QSR 7, were identified; their production occurred preferentially at high population density, required both LuxR and 3-oxo-C6-HSL, and was inhibited by C8-HSL at low population density. The genes encoding two of the QSR proteins were characterized: qsrP directs cells to synthesize an apparently novel periplasmic protein, and ribB is a homolog of the Escherichia coli gene for 3,4-dihydroxy-2-butanone 4-phosphate synthase, a key enzyme for riboflavin synthesis. The qsrP and ribB promoter regions each contained a sequence similar to the lux operon lux box, a 20-bp region of dyad symmetry necessary for LuxR/3-oxo-C6-HSL-dependent activation of lux operon transcription. V. fischeri qsrP and ribB mutants exhibited no distinct phenotype in culture. However, a qsrP mutant, in competition with its parent strain, was less successful in colonizing Euprymna scolopes, the symbiotic host of V. fischeri. The newly identified QSR genes, together with the lux operon, define a LuxR/acyl-HSL-responsive quorum-sensing regulon in V. fischeri.

  20. Histones and DNA Compete for Binding Polyphosphoinositides in Bilayers

    PubMed Central

    Lete, Marta G.; Sot, Jesús; Ahyayauch, Hasna; Fernández-Rivero, Noelia; Prado, Adelina; Goñi, Félix M.; Alonso, Alicia

    2014-01-01

    Recent discoveries on the presence and location of phosphoinositides in the eukaryotic cell nucleoplasm and nuclear membrane prompted us to study the putative interaction of chromatin components with these lipids in model membranes (liposomes). Turbidimetric studies revealed that a variety of histones and histone combinations (H1, H2AH2B, H3H4, octamers) caused a dose-dependent aggregation of phosphatidylcholine vesicles (large unilamellar vesicle or small unilamellar vesicle) containing negatively charged phospholipids. 5 mol % phosphatidylinositol-4-phosphate (PIP) was enough to cause extensive aggregation under our conditions, whereas with phosphatidylinositol (PI) at least 20 mol % was necessary to obtain a similar effect. Histone binding to giant unilamellar vesicle and vesicle aggregation was visualized by confocal microscopy. Histone did not cause vesicle aggregation in the presence of DNA, and the latter was able to disassemble the histone-vesicle aggregates. At DNA/H1 weight ratios 0.1–0.5 DNA- and PIP-bound H1 appear to coexist. Isothermal calorimetry studies revealed that the PIP-H1 association constant was one order of magnitude higher than that of PI-H1, and the corresponding lipid/histone stoichiometries were ∼0.5 and ∼1, respectively. The results suggest that, in the nucleoplasm, a complex interplay of histones, DNA, and phosphoinositides may be taking place, particularly at the nucleoplasmic reticula that reach deep within the nucleoplasm, or during somatic and nonsomatic nuclear envelope assembly. The data described here provide a minimal model for analyzing and understanding the mechanism of these interactions. PMID:24606933

  1. Phosphorus-assisted biomass thermal conversion: reducing carbon loss and improving biochar stability.

    PubMed

    Zhao, Ling; Cao, Xinde; Zheng, Wei; Kan, Yue

    2014-01-01

    There is often over 50% carbon loss during the thermal conversion of biomass into biochar, leading to it controversy for the biochar formation as a carbon sequestration strategy. Sometimes the biochar also seems not to be stable enough due to physical, chemical, and biological reactions in soils. In this study, three phosphorus-bearing materials, H3PO4, phosphate rock tailing (PRT), and triple superphosphate (TSP), were used as additives to wheat straw with a ratio of 1: 0.4-0.8 for biochar production at 500°C, aiming to alleviate carbon loss during pyrolysis and to increase biochar-C stabilization. All these additives remarkably increased the biochar yield from 31.7% (unmodified biochar) to 46.9%-56.9% (modified biochars). Carbon loss during pyrolysis was reduced from 51.7% to 35.5%-47.7%. Thermogravimetric analysis curves showed that the additives had no effect on thermal stability of biochar but did enhance its oxidative stability. Microbial mineralization was obviously reduced in the modified biochar, especially in the TSP-BC, in which the total CO2 emission during 60-d incubation was reduced by 67.8%, compared to the unmodified biochar. Enhancement of carbon retention and biochar stability was probably due to the formation of meta-phosphate or C-O-PO3, which could either form a physical layer to hinder the contact of C with O2 and bacteria, or occupy the active sites of the C band. Our results indicate that pre-treating biomass with phosphors-bearing materials is effective for reducing carbon loss during pyrolysis and for increasing biochar stabilization, which provides a novel method by which biochar can be designed to improve the carbon sequestration capacity.

  2. Immobilization of lead in anthropogenic contaminated soils using phosphates with/without oxalic acid.

    PubMed

    Su, Xiaojuan; Zhu, Jun; Fu, Qingling; Zuo, Jichao; Liu, Yonghong; Hu, Hongqing

    2015-02-01

    Understanding the effects of oxalic acid (OA) on the immobilization of Pb(II) in contaminated soils by phosphate materials, has considerable benefits for risk assessment and remediation strategies for the soil. A series of phosphate amendments with/without oxalic acid were applied to two anthropogenic contaminated soils. We investigated the immobilization of Pb(II) by KH2PO4, phosphate rock (PR), activated phosphate rock (APR) and synthetic hydroxyapatite (HAP) at different phosphate:Pb (P:Pb) molar ratios (0, 0.6, 2.0 and 4.0) in the presence/absence of 50 mmol oxalic acid/kg soil, respectively. The effects of treatments were evaluated using single extraction with deionized water or CaCl2, Community Bureau of Reference (BCR) sequential extraction and toxicity characteristic leaching procedure (TCLP) methods. Our results showed that the concentration of water extractable, exchangeable and TCLP-Pb all decreased with incubation time. The concentration of water-extractable Pb after 120 days was reduced by 100% when soils were amended with APR, HAP and HAP+OA, and the TCLP-Pb was <5 mg/L for the red soil at P:Pb molar ratio 4.0. Water-soluble Pb could not be detected and the TCLP-Pb was <5 mg/L at all treatments applied to the yellow-brown soil. BCR results indicated that APR was most effective, although a slight enhancement of water-soluble phosphate was detected at the P:Pb molar ratio 4.0 at the beginning of incubation. Oxalic acid activated phosphates, and so mixing insoluble phosphates with oxalic acid may be a useful strategy to improve their effectiveness in reducing Pb bioavailability.

  3. Crystal structure of a sodium, zinc and iron(III)-based non-stoichiometric phosphate with an alluaudite-like structure: Na1.67Zn1.67Fe1.33(PO4)3.

    PubMed

    Khmiyas, Jamal; Assani, Abderrazzak; Saadi, Mohamed; El Ammari, Lahcen

    2015-06-01

    The new title compound, disodium dizinc iron(III) tris-(phosphate), Na1.67Zn1.67Fe1.33(PO4)3, which belongs to the alluaudite family, has been synthesized by solid-state reactions. In this structure, all atoms are in general positions except for four, which are located on special positions of the C2/c space group. This structure is characterized by cation substitutional disorder at two sites, one situated on the special position 4e (2) and the other on the general position 8f. The 4e site is partially occupied by Na(+) [0.332 (3)], whereas the 8f site is entirely filled by a mixture of Fe and Zn. The full-occupancy sodium and zinc atoms are located at the Wyckoff positions on the inversion center 4a (-1) and on the twofold rotation axis 4e, respectively. Refinement of the occupancy ratios, bond-valence analysis and the electrical neutrality requirement of the structure lead to the given composition for the title compound. The three-dimensional framework of this structure consists of kinked chains of edge-sharing octa-hedra stacked parallel to [10-1]. The chains are formed by a succession of trimers based on [ZnO6] octa-hedra and the mixed-cation Fe(III)/Zn(II) [(Fe/Zn)O6] octa-hedra [Fe(III):Zn(III) ratio 0.668 (3)/0.332 (3)]. Continuous chains are held together by PO4 phosphate groups, forming polyhedral sheets perpendicular to [010]. The stacked sheets delimit two types of tunnels parallel to the c axis in which the sodium cations are located. Each Na(+) cation is coordinated by eight O atoms. The disorder of Na in the tunnel might presage ionic mobility for this material.

  4. Synthesis and characterization of insulin/zirconium phosphate@TiO2 hybrid composites for enhanced oral insulin delivery applications.

    PubMed

    Safari, Mostafa; Kamari, Younes; Ghiaci, Mehran; Sadeghi-Aliabadi, Hojjat; Mirian, Mina

    2017-05-01

    In this work, a series of composites of insulin (Ins)/zirconium phosphate (ZrP) were synthesized by intercalation method, then, these composites were coated with TiO2 by sol-gel method to prepare Ins/ZrP@TiO2 hybrid composites and the drug release of the composites was investigated by using UV-Vis spectroscopy. Ins/ZrP (10, 30, 60 wt%) composites were prepared by intercalation of insulin into the ZrP layers in water. Then Ins/ZrP composites were coated with different amounts of TiO2 (30, 50, 100 wt %) by using titanium tetra n-butoxide, as precursor. Formation of intercalated Ins/ZrP and Ins/ZrP@TiO2 hybrid composites was characterized by FT-IR, FE-SEM, BET and XRD analysis. Zeta potential of the optimized Ins/ZrP@TiO2 hybrid composite was determined -27.2 mV. Cytotoxic effects of the optimized Ins/ZrP@TiO2 hybrid composite against HeLa and Hek293T cell lines were evaluated using MTT assay and the results showed that designed drug delivery system was not toxic in biological environment. Compared to the Ins/ZrP composites, incorporation of TiO2 coating enhanced the drug entrapment considerably, and reduced the drug release. The Ins/ZrP composites without TiO2 coating released the whole drug after 30 min in pH 7.4 (phosphate buffer solution) while the TiO2-coated composites released the entrapped drug after 20 h. In addition to increasing the shelf life of hormone, this nanoencapsulation and nanocoating method can convert the insulin utilization from injection to oral and present a painless and more comfortable treatment for diabetics.

  5. Membraneless glucose/oxygen enzymatic fuel cells using redox hydrogel films containing carbon nanotubes.

    PubMed

    MacAodha, Domhnall; Ó Conghaile, Peter; Egan, Brenda; Kavanagh, Paul; Leech, Dónal

    2013-07-22

    Co-immobilisation of three separate multiple blue copper oxygenases, a Myceliophthora thermophila laccase, a Streptomyces coelicolor laccase and a Myrothecium verrucaria bilirubin oxidase, with an [Os(2,2'-bipyridine)2 (polyvinylimidazole)10Cl](+/2+) redox polymer in the presence of multi-walled carbon nanotubes (MWCNTs) on graphite electrodes results in enzyme electrodes that produce current densities above 0.5 mA cm(-2) for oxygen reduction at an applied potential of 0 V versus Ag/AgCl. Fully enzymatic membraneless fuel cells are assembled with the oxygen-reducing enzyme electrodes connected to glucose-oxidising anodes based on co-immobilisation of glucose oxidase or a flavin adenine dinucleotide-dependent glucose dehydrogenase with an [Os(4,4'-dimethyl-2,2'-bipyridine)2(polyvinylimidazole)10Cl](+/2+) redox polymer in the presence of MWCNTs on graphite electrodes. These fuel cells can produce power densities of up to 145 μW cm(-2) on operation in pH 7.4 phosphate buffer solution at 37 °C containing 150 mM NaCl, 5 mM glucose and 0.12 mM O2. The fuel cells based on Myceliophthora thermophila laccase enzyme electrodes produce the highest power density if combined with glucose oxidase-based anodes. Although the maximum power density of a fuel cell of glucose dehydrogenase and Myceliophthora thermophila laccase enzyme electrodes decreases from 110 μW cm(-2) in buffer to 60 μW cm(-2) on testing in artificial plasma, it provides the highest power output reported to date for a fully enzymatic glucose-oxidising, oxygen-reducing fuel cell in artificial plasma.

  6. Over-expression of DXS gene enhances terpenoidal secondary metabolite accumulation in rose-scented geranium and Withania somnifera: active involvement of plastid isoprenogenic pathway in their biosynthesis.

    PubMed

    Jadaun, Jyoti Singh; Sangwan, Neelam S; Narnoliya, Lokesh K; Singh, Neha; Bansal, Shilpi; Mishra, Bhawana; Sangwan, Rajender Singh

    2017-04-01

    Rose-scented geranium (Pelargonium spp.) is one of the most important aromatic plants and is well known for its diverse perfumery uses. Its economic importance is due to presence of fragrance rich essential oil in its foliage. The essential oil is a mixture of various volatile phytochemicals which are mainly terpenes (isoprenoids) in nature. In this study, on the geranium foliage genes related to isoprenoid biosynthesis (DXS, DXR and HMGR) were isolated, cloned and confirmed by sequencing. Further, the first gene of 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway, 1-deoxy-d-xylulose-5-phosphate synthase (GrDXS), was made full length by using rapid amplification of cDNA ends strategy. GrDXS contained a 2157 bp open reading frame that encoded a polypeptide of 792 amino acids having calculated molecular weight 77.5 kDa. This study is first report on heterologous expression and kinetic characterization of any gene from this economically important plant. Expression analysis of these genes was performed in different tissues as well as at different developmental stages of leaves. In response to external elicitors, such as methyl jasmonate, salicylic acid, light and wounding, all the three genes showed differential expression profiles. Further GrDXS was over expressed in the homologous (rose-scented geranium) as well as in heterologous (Withania somnifera) plant systems through genetic transformation approach. The over-expression of GrDXS led to enhanced secondary metabolites production (i.e. essential oil in rose-scented geranium and withanolides in W. somnifera). To the best of our knowledge, this is the first report showing the expression profile of the three genes related to isoprenoid biosynthesis pathways operated in rose-scented geranium as well as functional characterization study of any gene from rose-scented geranium through a genetic transformation system.

  7. Metabolic network reconstruction, growth characterization and 13C-metabolic flux analysis of the extremophile Thermus thermophilus HB8.

    PubMed

    Swarup, Aditi; Lu, Jing; DeWoody, Kathleen C; Antoniewicz, Maciek R

    2014-07-01

    Thermus thermophilus is an extremely thermophilic bacterium with significant biotechnological potential. In this work, we have characterized aerobic growth characteristics of T. thermophilus HB8 at temperatures between 50 and 85°C, constructed a metabolic network model of its central carbon metabolism and validated the model using (13)C-metabolic flux analysis ((13)C-MFA). First, cells were grown in batch cultures in custom constructed mini-bioreactors at different temperatures to determine optimal growth conditions. The optimal temperature for T. thermophilus grown on defined medium with glucose was 81°C. The maximum growth rate was 0.25h(-1). Between 50 and 81°C the growth rate increased by 7-fold and the temperature dependence was described well by an Arrhenius model with an activation energy of 47kJ/mol. Next, we performed a (13)C-labeling experiment with [1,2-(13)C] glucose as the tracer and calculated intracellular metabolic fluxes using (13)C-MFA. The results provided support for the constructed network model and highlighted several interesting characteristics of T. thermophilus metabolism. We found that T. thermophilus largely uses glycolysis and TCA cycle to produce biosynthetic precursors, ATP and reducing equivalents needed for cells growth. Consistent with its proposed metabolic network model, we did not detect any oxidative pentose phosphate pathway flux or Entner-Doudoroff pathway activity. The biomass precursors erythrose-4-phosphate and ribose-5-phosphate were produced via the non-oxidative pentose phosphate pathway, and largely via transketolase, with little contribution from transaldolase. The high biomass yield on glucose that was measured experimentally was also confirmed independently by (13)C-MFA. The results presented here provide a solid foundation for future studies of T. thermophilus and its metabolic engineering applications.

  8. Synthesis, separation, identification and interconversion of riboflavin phosphates and their acetyl derivatives: a reinvestigation.

    PubMed

    Scola-Nagelschneider, G; Hemmerich, P

    1976-07-15

    A reinvestigation of flavin phosphate synthesis, separation, identification, and interconversion was made in view of contradictory results in the literature. It has been confirmed that monochlorophosphoric acid is the best agent for selective 5'-monophosphorylation of riboflavin and derivatives. This reaction yields, however, invariably up to 20% of an isomer, which has been separated by preparative thick-layer chromatography and shown to be the 4'-monophosphate. All the earlier authors failed to detect this isomer which does not bind to flavodoxin. It equilibrates in dilute mineral acid to yield an 8:2 mixture of 5'-phosphate to 4'-phosphate by phosphate migration. The formation of 2',3',4'-triacetyl-flavin mononucleotide, according to Christie, S. M. H., Kenner, G. W. & Todd, A. R. (1954) J. Chem. Soc., 46-52, upon acid-catalysed acetylation of pure FMN, was confirmed. The same reaction under base catalysis, however, does not yield 2',3',4'-triacetyl-flavin mononucleotide as claimed by Khomutova, E. D., Shapiro, T. A., Mezentseva, M. W. & Berezovskii, V. M. (1965) Otd. Obshch. i. Tekhn. Khim., 241-244, Chem. Abstr. 65, 5516a, but in fact up to 80% 2', 3'-diacetyl-flavin 4':5'-cyclophosphate as the main product, which is stable under neutral and weak acidic conditions and does not hydrolyse to 2',3'-diacetyl-flavin 5'-monophosphate as claimed by McCormick, D.B. (1974) J. Heterocycl. Chem. 11, 969-974. The various flavin phosphates and their acetyl derivatives have been identified by pH-titration, electrophoresis, and proton magnetic resonance spectrometry, which direct analyses of crude reaction products as well as a rapid purity check of commerical FMN.

  9. Expression of 3-hydroxy-3-methylglutaryl-CoA reductase, p-hydroxybenzoate-m-geranyltransferase and genes of phenylpropanoid pathway exhibits positive correlation with shikonins content in arnebia [Arnebia euchroma (Royle) Johnston

    PubMed Central

    2010-01-01

    Background Geranyl pyrophosphate (GPP) and p-hydroxybenzoate (PHB) are the basic precursors involved in shikonins biosynthesis. GPP is derived from mevalonate (MVA) and/or 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway(s), depending upon the metabolite and the plant system under consideration. PHB, however, is synthesized by only phenylpropanoid (PP) pathway. GPP and PHB are central moieties to yield shikonins through the synthesis of m-geranyl-p-hydroxybenzoate (GHB). Enzyme p-hydroxybenzoate-m-geranyltransferase (PGT) catalyses the coupling of GPP and PHB to yield GHB. The present research was carried out in shikonins yielding plant arnebia [Arnebia euchroma (Royle) Johnston], wherein no molecular work has been reported so far. The objective of the work was to identify the preferred GPP synthesizing pathway for shikonins biosynthesis, and to determine the regulatory genes involved in the biosynthesis of GPP, PHB and GHB. Results A cell suspension culture-based, low and high shikonins production systems were developed to facilitate pathway identification and finding the regulatory gene. Studies with mevinolin and fosmidomycin, inhibitors of MVA and MEP pathway, respectively suggested MVA as a preferred route of GPP supply for shikonins biosynthesis in arnebia. Accordingly, genes of MVA pathway (eight genes), PP pathway (three genes), and GHB biosynthesis were cloned. Expression studies showed down-regulation of all the genes in response to mevinolin treatment, whereas gene expression was not influenced by fosmidomycin. Expression of all the twelve genes vis-à-vis shikonins content in low and high shikonins production system, over a period of twelve days at frequent intervals, identified critical genes of shikonins biosynthesis in arnebia. Conclusion A positive correlation between shikonins content and expression of 3-hydroxy-3-methylglutaryl-CoA reductase (AeHMGR) and AePGT suggested critical role played by these genes in shikonins biosynthesis. Higher

  10. Effector Vγ9Vδ2 T cells dominate the human fetal γδ T-cell repertoire.

    PubMed

    Dimova, Tanya; Brouwer, Margreet; Gosselin, Françoise; Tassignon, Joël; Leo, Oberdan; Donner, Catherine; Marchant, Arnaud; Vermijlen, David

    2015-02-10

    γδ T cells are unconventional T cells recognizing antigens via their γδ T-cell receptor (TCR) in a way that is fundamentally different from conventional αβ T cells. γδ T cells usually are divided into subsets according the type of Vγ and/or Vδ chain they express in their TCR. T cells expressing the TCR containing the γ-chain variable region 9 and the δ-chain variable region 2 (Vγ9Vδ2 T cells) are the predominant γδ T-cell subset in human adult peripheral blood. The current thought is that this predominance is the result of the postnatal expansion of cells expressing particular complementary-determining region 3 (CDR3) in response to encounters with microbes, especially those generating phosphoantigens derived from the 2-C-methyl-d-erythritol 4-phosphate pathway of isoprenoid synthesis. However, here we show that, rather than requiring postnatal microbial exposure, Vγ9Vδ2 T cells are the predominant blood subset in the second-trimester fetus, whereas Vδ1(+) and Vδ3(+) γδ T cells are present only at low frequencies at this gestational time. Fetal blood Vγ9Vδ2 T cells are phosphoantigen responsive and display very limited diversity in the CDR3 of the Vγ9 chain gene, where a germline-encoded sequence accounts for >50% of all sequences, in association with a prototypic CDR3δ2. Furthermore, these fetal blood Vγ9Vδ2 T cells are functionally preprogrammed (e.g., IFN-γ and granzymes-A/K), with properties of rapidly activatable innatelike T cells. Thus, enrichment for phosphoantigen-responsive effector T cells has occurred within the fetus before postnatal microbial exposure. These various characteristics have been linked in the mouse to the action of selecting elements and would establish a much stronger parallel between human and murine γδ T cells than is usually articulated.

  11. Long-term atmospheric nutrient inputs to the Eastern Mediterranean: sources, solubility and comparison with riverine inputs

    NASA Astrophysics Data System (ADS)

    Koçak, M.; Kubilay, N.; Tuǧrul, S.; Mihalopoulos, N.

    2010-07-01

    Aerosol and rain samples were collected at a rural site located on the coastline of the Eastern Mediterranean, Erdemli, Turkey between January 1999 and December 2007. Riverine sampling was carried out at five Rivers (Ceyhan, Seyhan, Göksu, Berdan and Lamas) draining into the Northeastern Levantine Basin (NLB) between March 2002 and July 2007. Samples were analyzed for macronutrients of phosphate, silicate, nitrate and ammonium (PO43-, Sidiss, NO3- and NH4+). Phosphate and silicate in aerosol and rainwater showed higher and larger variation during the transitional period (March-May, September) when air flows predominantly originate from North Africa and Middle East/Arabian Peninsula. Deficiency of alkaline material were found to be the main reason of the acidic rain events whilst high pH values (>7) were associated with high Sidiss concentrations due to sporadic dust events. In general, lowest nitrate and ammonium concentrations in aerosol and rainwater were associated with air flow from the Mediterranean Sea. Unlike NO3- and NH4+ (Dissolved Inorganic Nitrogen, DIN), there were statistical differences for PO43- and Sidiss solubilities in sea-water and pure-water. Solubilities of PO43- and Sidiss were found to be related with air mass back trajectories and pH. Comparison of atmospheric with riverine fluxes demonstrated that DIN and PO43- fluxes to NLB were dominated by atmosphere (~90% and ~60% respectively) whereas the input of Si was mainly derived from riverine runoff (~90%). N/P ratios (atmosphere ~233; riverine ~28) revealed that NLB receives excessive amounts of DIN and this unbalanced P and N inputs may provoke even more phosphorus deficiency. Molar Si/N ratios (atmosphere + riverine) suggested Si limitation which might cause a switch from diatom dominated phytoplankton communities to non-siliceous populations in NLB.

  12. Phospholipase C mediates (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI)-, but not lysergic acid diethylamide (LSD)-elicited head bobs in rabbit medial prefrontal cortex.

    PubMed

    Schindler, Emmanuelle A D; Harvey, John A; Aloyo, Vincent J

    2013-01-23

    The phenethylamine and indoleamine classes of hallucinogens demonstrate distinct pharmacological properties, although they share a serotonin(2A) (5-HT(2A)) receptor mechanism of action (MOA). The 5-HT(2A) receptor signals through phosphatidylinositol (PI) hydrolysis, which is initiated upon activation of phospholipase C (PLC). The role of PI hydrolysis in the effects of hallucinogens remains unclear. In order to better understand the role of PI hydrolysis in the MOA of hallucinogens, the PLC inhibitor, 1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U73122), was used to study the effects of two hallucinogens, the phenethylamine, (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), and the indoleamine, lysergic acid diethylamide (LSD). PI hydrolysis was quantified through release of [3H]inositol-4-phosphate from living rabbit frontocortical tissue prisms. Head bobs were counted after hallucinogens were infused into the medial prefrontal cortex (mPFC) of rabbits. Both DOI and LSD stimulated PI hydrolysis in frontocortical tissue through activation of PLC. DOI-stimulated PI hydrolysis was blocked by 5-HT(2A/2C) receptor antagonist, ketanserin, whereas the LSD signal was blocked by 5-HT(2B/2C) receptor antagonist, SB206553. When infused into the mPFC, both DOI- and LSD-elicited head bobs. Pretreatment with U73122 blocked DOI-, but not LSD-elicited head bobs. The two hallucinogens investigated were distinct in their activation of the PI hydrolysis signaling pathway. The serotonergic receptors involved with DOI and LSD signals in frontocortical tissue were different. Furthermore, PLC activation in mPFC was necessary for DOI-elicited head bobs, whereas LSD-elicited head bobs were independent of this pathway. These novel findings urge closer investigation into the intracellular mechanism of action of these unique compounds.

  13. Biosynthesis of sesquiterpenes in grape berry exocarp of Vitis vinifera L.: evidence for a transport of farnesyl diphosphate precursors from plastids to the cytosol

    PubMed Central

    May, Bianca; Lange, B. Markus; Wüst, Matthias

    2013-01-01

    The participation of the mevalonic acid (MVA) and 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol-4-phosphate (DOXP/MEP) pathways in sesquiterpene biosynthesis of grape berries was investigated. There is an increasing interest in this class of terpenoids, since the oxygenated sesquiterpene rotundone was identified as the peppery aroma impact compound in Australian Shiraz wines. To investigate precursor supply pathway utilization, in vivo feeding experiments were performed with the deuterium labeled, pathway specific, precursors [5,5-2H2]-1-deoxy-D-xylulose and [5,5-2H2]-mevalonic acid lactone. Head Space-Solid Phase Micro Extraction-Gas Chromatography-Mass Spectrometry (HS-SPME-GC-MS) analysis of the generated volatile metabolites demonstrated that de novo sesquiterpene biosynthesis is mainly located in the grape berry exocarp (skin), with no detectable activity in the mesocarp (flesh) of the Lemberger variety. Interestingly, precursors from both the (primarily) cytosolic MVA and plastidial DOXP/MEP pathways were incorporated into grape sesquiterpenes in the varieties Lemberger, Gewürztraminer and Syrah. Our labeling data provide evidence for a homogenous, cytosolic pool of precursors for sesquiterpene biosynthesis, indicating that a transport of precursors occurs mostly from plastids to the cytosol. The labeling patterns of the sesquiterpene germacrene D were in agreement with a cyclization mechanism analogous to that of a previously cloned enantioselective (R)-germacrene D synthase from Solidago canadensis. This observation was subsequently confirmed by enantioselective GC-MS analysis demonstrating the exclusive presence of (R)-germacrene D, and not the (S)-enantiomer, in grape berries. PMID:23954075

  14. Interleukin 1 stimulates phosphatidylinositol kinase activity in human fibroblasts.

    PubMed Central

    Ballou, L R; Barker, S C; Postlethwaite, A E; Kang, A H

    1991-01-01

    IL-1 mediates multiple cellular immune and inflammatory responses, but little is known of the intracellular biochemical mechanisms involved in IL-1 actions. We studied the effects of IL-1 on phosphatidylinositol (PtdIns) metabolism and confirmed reports indicating that IL-1 does not stimulate increased PtdIns turnover; however, we observed the accumulation of PtdIns-4-phosphate (PtdInsP) in response to IL-1. Using a fibroblast membrane preparation, we were able to detect stimulated PtdInsP accumulation within 10 s of IL-1 addition. Increased PtdInsP accumulation was due to stimulated PtdIns kinase activity, not the inhibition of PtdInsP hydrolysis by phospholipase(s). PtdIns kinase activity was magnesium dependent, increased as a function of IL-1 concentration, and specifically phosphorylated the D4 position of inositol. Stimulated PtdIns kinase activity could be detected at 10(-12) M IL-1 in fibroblast membranes, a concentration within the physiological range for IL-1 action; half-maximal activity was reached at approximately 10(-10) M IL-1. Heat denaturation of IL-1 or treatment of IL-1 with anti-IL-1 antibody abrogated the IL-1 effect. These findings demonstrate the direct, IL-1-mediated, stimulation of PtdIns kinase. IL-1-stimulated PtdIns kinase activity represents an important physiological regulatory effect by IL-1 as it could control the synthesis and/or maintenance of phosphorylated derivatives of PtdIns which comprise only a very small pool of substrates for the generation of the second messengers inositol 1,4,5-triphosphate and diacylglycerol. PMID:1845871

  15. A randomized Phase II trial of the tumor vascular disrupting agent CA4P (fosbretabulin tromethamine) with carboplatin, paclitaxel, and bevacizumab in advanced nonsquamous non-small-cell lung cancer

    PubMed Central

    Garon, Edward B; Neidhart, Jeffrey D; Gabrail, Nashat Y; de Oliveira, Moacyr R; Balkissoon, Jai; Kabbinavar, Fairooz

    2016-01-01

    Introduction Combretastatin A4-phosphate, fosbretabulin tromethamine (CA4P) is a vascular disrupting agent that targets tumor vasculature. This study evaluated the safety of CA4P when combined with carboplatin, paclitaxel, and bevacizumab in chemotherapy-naïve subjects with advanced nonsquamous, non-small-cell lung cancer. Methods Adult subjects with confirmed American Joint Committee on Cancer six stage IIIB/IV non-small-cell lung cancer and an Eastern Cooperative Oncology Group performance score of 0 or 1 were randomized to receive six cycles (treatment phase) of paclitaxel (200 mg/m2), carboplatin (area under the concentration versus time curve 6), and bevacizumab (15 mg/kg) on day 1 and repeated every 21 days, or this regimen plus CA4P (60 mg/m2) on days 7, 14, and 21 of each cycle. Subjects could then receive additional maintenance treatment (excluding carboplatin and paclitaxel) for up to 1 year. Results Sixty-three subjects were randomized, 31 to control and 32 to CA4P, and 19 (61.3%) and 17 (53.1%), respectively, completed the treatment phase. Exposure to study treatment and dose modifications were comparable between the randomized groups. The overall incidence of treatment-emergent adverse events was similar between groups, with increased neutropenia, leukopenia, and hypertension in the CA4P group. Deaths, serious adverse events, and early discontinuations from treatment were comparable between the randomized treatment groups. The overall tumor response rate with CA4P was 50% versus 32% in controls. Overall and progression-free survival rates were comparable between the groups. Conclusion CA4P plus carboplatin, paclitaxel, and bevacizumab appears to be a tolerable regimen with an acceptable toxicity profile in subjects with advanced non-small-cell lung cancer. PMID:27942221

  16. Alteration of the flexible loop in 1-deoxy-D-xylulose-5-phosphate reductoisomerase boosts enthalpy-driven inhibition by fosmidomycin.

    PubMed

    Kholodar, Svetlana A; Tombline, Gregory; Liu, Juan; Tan, Zhesen; Allen, C Leigh; Gulick, Andrew M; Murkin, Andrew S

    2014-06-03

    1-Deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), which catalyzes the first committed step in the 2-C-methyl-d-erythritol 4-phosphate pathway of isoprenoid biosynthesis used by Mycobacterium tuberculosis and other infectious microorganisms, is absent in humans and therefore an attractive drug target. Fosmidomycin is a nanomolar inhibitor of DXR, but despite great efforts, few analogues with comparable potency have been developed. DXR contains a strictly conserved residue, Trp203, within a flexible loop that closes over and interacts with the bound inhibitor. We report that while mutation to Ala or Gly abolishes activity, mutation to Phe and Tyr only modestly impacts kcat and Km. Moreover, pre-steady-state kinetics and primary deuterium kinetic isotope effects indicate that while turnover is largely limited by product release for the wild-type enzyme, chemistry is significantly more rate-limiting for W203F and W203Y. Surprisingly, these mutants are more sensitive to inhibition by fosmidomycin, resulting in Km/Ki ratios up to 19-fold higher than that of wild-type DXR. In agreement, isothermal titration calorimetry revealed that fosmidomycin binds up to 11-fold more tightly to these mutants. Most strikingly, mutation strongly tips the entropy-enthalpy balance of total binding energy from 50% to 75% and 91% enthalpy in W203F and W203Y, respectively. X-ray crystal structures suggest that these enthalpy differences may be linked to differences in hydrogen bond interactions involving a water network connecting fosmidomycin's phosphonate group to the protein. These results confirm the importance of the flexible loop, in particular Trp203, in ligand binding and suggest that improved inhibitor affinity may be obtained against the wild-type protein by introducing interactions with this loop and/or the surrounding structured water network.

  17. Dynamics of renal electrolyte excretion in growing mice.

    PubMed

    Schmidt, Katharina; Ripper, Maria; Tegtmeier, Ines; Humberg, Evelyn; Sterner, Christina; Reichold, Markus; Warth, Richard; Bandulik, Sascha

    2013-01-01

    Genetically modified mice represent important models for elucidating renal pathophysiology, but gene deletions frequently cause severe failure to thrive. In such cases, the analysis of the phenotype is often limited to the first weeks of life when renal excretory function undergoes dramatic physiological changes. Here, we investigated the postnatal dynamics of urinary ion excretion in mice. The profiles of urinary electrolyte excretion of mice were examined from birth until after weaning using an automated ion chromatography system. Postnatally, mice grew about 0.4 g/day, except during two phases with slower weight gain: (i) directly after birth during adaptation to extrauterine conditions (P0-P2) and (ii) during the weaning period (P15-P21), when nutrition changed from mother's milk to solid chow and water. During the first 3 days after birth, remarkable changes in urinary Na(+), Ca(2+), Mg(2+), and phosphate concentrations occurred, whereas K(+) and Cl(-) concentrations hardly changed. From days 4-14 after birth, Na(+), Ca(2+), Mg(2+), K(+), and Cl(-) concentrations remained relatively stable at low levels. Urinary concentrations of creatinine, NH4(+), phosphate, and sulfate constantly increased from birth until after weaning. Profiles of salt excretion in KCNJ10(-/-) mice exemplified the relevance of age-dependent analysis of urinary excretion. In conclusion, the most critical phases for analysis of renal ion excretion during the first weeks of life are directly after birth and during the weaning period. The age dependence of urinary excretion varies for the different ions. This should be taken into consideration when the renal phenotype of mice is investigated during the first weeks of life.

  18. Contribution of G-CSF to the acute mobilization of endothelial precursor cells by vascular disrupting agents

    PubMed Central

    Shaked, Yuval; Tang, Terence; Woloszynek, Jill; Daenen, Laura G.; Man, Shan; Xu, Ping; Cai, Shi-Rong; Arbeit, Jeffrey M.; Voest, Emile E.; Chaplin, David; Smythe, Jon; Harris, Adrian; Nathan, Paul; Judson, Ian; Rustin, Gordon; Bertolini, Francesco; Link, Daniel C.; Kerbel, Robert S.

    2009-01-01

    Vascular disrupting agents (VDAs) cause acute shutdown of abnormal established tumor vasculature, followed by massive intratumoral hypoxia and necrosis. However, a viable rim of tumor tissue invariably remains from which tumor regrowth rapidly resumes. We have recently shown that an acute systemic mobilization and homing of bone marrow derived circulating endothelial precursor cells (CEPs) can promote tumor regrowth following treatment with either a VDA or certain chemotherapy drugs. The molecular mediators of this systemic reactive host process are unknown. Here we show that following treatment of mice with OXi-4503, a second generation potent pro-drug derivative of combretastatin-A 4 phosphate (CA4P), rapid increases in circulating plasma VEGF, SDF-1, and G-CSF levels are detected. With the aim of determining whether G-CSF is involved in VDA-induced CEP mobilization, mutant G-CSF-R−/− mice were treated with OXI-4503. We found that as opposed to wildtype controls, G-CSF-R−/− mice failed to mobilize CEPs or show induction of SDF-1 plasma levels. Furthermore, Lewis lung carcinomas grown in such mice treated with OXi-4503 showed greater levels of necrosis compared to tumors treated in wildtype mice. Evidence for rapid elevations in circulating plasma G-CSF, VEGF, and SDF-1 were also observed in VDA (CA4P) treated cancer patients. These results highlight the possible impact of drug-induced G-CSF on tumor re-growth following certain cytotoxic drug therapies, in this case using a VDA, and hence G-CSF as a possible therapeutic target. PMID:19738066

  19. Expression cloning and biochemical characterization of a Rhizobium leguminosarum lipid A 1-phosphatase.

    PubMed

    Karbarz, Mark J; Kalb, Suzanne R; Cotter, Robert J; Raetz, Christian R H

    2003-10-10

    Lipid A of Rhizobium leguminosarum, a nitrogen-fixing plant endosymbiont, displays several significant structural differences when compared with Escherichia coli. An especially striking feature of R. leguminosarum lipid A is that it lacks both the 1- and 4'-phosphate groups. Distinct lipid A phosphatases that attack either the 1 or the 4' positions have previously been identified in extracts of R. leguminosarum and Rhizobium etli but not Sinorhizobium meliloti or E. coli. Here we describe the identification of a hybrid cosmid (pMJK-1) containing a 25-kb R. leguminosarum 3841 DNA insert that directs the overexpression of the lipid A 1-phosphatase. Transfer of pMJK-1 into S. meliloti 1021 results in heterologous expression of 1-phosphatase activity, which is normally absent in extracts of strain 1021, and confers resistance to polymyxin. Sequencing of a 7-kb DNA fragment derived from the insert of pMJK-1 revealed the presence of a lipid phosphatase ortholog (designated LpxE). Expression of lpxE in E. coli behind the T7lac promoter results in the appearance of robust 1-phosphatase activity, which is normally absent in E. coli membranes. Matrix-assisted laser-desorption/time of flight and radiochemical analysis of the product generated in vitro from the model substrate lipid IVA confirms the selective removal of the 1-phosphate group. These findings show that lpxE is the structural gene for the 1-phosphatase. The availability of lpxE may facilitate the re-engineering of lipid A structures in diverse Gram-negative bacteria and allow assessment of the role of the 1-phosphatase in R. leguminosarum symbiosis with plants. Possible orthologs of LpxE are present in some intracellular human pathogens, including Francisella tularensis, Brucella melitensis, and Legionella pneumophila.

  20. Signal transduction in normal and pathological thrombin-stimulated human platelets.

    PubMed

    Rendu, F; Marche, P; Viret, J; Maclouf, J; Lebret, M; Tenza, D; Caen, J; Levy-Toledano, S

    1987-04-01

    Human blood platelets stimulated by thrombin undergo very rapid morphological changes, the most characteristic of which are pseudopod formation and granule centralization. These early changes in shape are accompanied by a transient decrease (30%) in phosphatidyl inositol 4,5-bisphosphate (PIP2) which occurs in the first 10 s after thrombin addition. Transient decreases in phosphatidyl inositol 4-phosphate (PIP) and phosphatidyl inositol (PI) occur later (20-30 s). These events lead to the formation of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DG) and hence phosphatidate (PA). Two polypeptides are phosphorylated during the same time span: the myosin light chain (P20) and a 43 kDa protein (P43). Concomitant with these molecular changes, platelet 'release reaction' occurs, i.e., liberation of the different granule constituents into the external medium: the earliest concerns dense bodies which liberate adenine nucleotides, calcium and serotonin; alpha-granules then liberate adhesive and specific proteins and are followed by lysosomes which liberate hydrolases. Pathological platelets from patients with inherited disorders, presenting well-characterized and specific defects of either the platelet membrane (GT) or storage granules (GPS and HPS), have also been studied. The results obtained lead to the following conclusions: (1) the transducing system is normal in platelets unable to aggregate; (2) phosphorylation of P20 and P43 proteins can be complete with impaired release; and (3) when platelets lack alpha-granules the transducing system as well as the release of other granule populations are impaired. These results evidence the relationship between the absence of intraplatelet components and metabolic events.

  1. Comprehensive assessment of the genes involved in withanolide biosynthesis from Withania somnifera: chemotype-specific and elicitor-responsive expression.

    PubMed

    Agarwal, Aditya Vikram; Gupta, Parul; Singh, Deeksha; Dhar, Yogeshwar Vikram; Chandra, Deepak; Trivedi, Prabodh Kumar

    2017-03-11

    Withania somnifera (L.) Dunal (Family, Solanaceae), is among the most valuable medicinal plants used in Ayurveda owing to its rich reservoir of pharmaceutically active secondary metabolites known as withanolides. Withanolides are C28-steroidal lactones having a triterpenoidal metabolic origin synthesised via mevalonate (MVA) pathway and methyl-D-erythritol-4-phosphate (MEP) pathway involving metabolic intermediacy of 24-methylene (C30-terpenoid) cholesterol. Phytochemical studies suggest differences in the content and/or nature of withanolides in different tissues of different chemotypes. Though development of genomic resources has provided information about putative genes encoding enzymes for biosynthesis of intermediate steps of terpenoid backbone, not much is known about their regulation and response to elicitation. In this study, we generated detailed molecular information about genes catalysing key regulatory steps of withanolide biosynthetic pathway. The full-length sequences of genes encoding enzymes for intermediate steps of terpenoid backbone biosynthesis and their paralogs have been characterized for their functional and structural properties as well as phylogeny using bioinformatics approach. The expression analysis suggests that these genes are differentially expressed in different tissues (with maximal expression in young leaf), chemotypes and in response to salicylic acid (SA) and methyl jasmonate (MJ) treatments. Sub-cellular localization studies suggest that both paralogs of sterol ∆-7 reductase (WsDWF5-1 and WsDWF5-2) are localized in the endoplasmic reticulum (ER) thus supporting their indispensible role in withanolide biosynthesis. Comprehensive information developed, in this study, will lead to elucidation of chemotype- as well as tissue-specific withanolide biosynthesis and development of new tools for functional genomics in this important medicinal plant.

  2. Legionella pneumophila Effector LpdA Is a Palmitoylated Phospholipase D Virulence Factor.

    PubMed

    Schroeder, Gunnar N; Aurass, Philipp; Oates, Clare V; Tate, Edward W; Hartland, Elizabeth L; Flieger, Antje; Frankel, Gad

    2015-10-01

    Legionella pneumophila is a bacterial pathogen that thrives in alveolar macrophages, causing a severe pneumonia. The virulence of L. pneumophila depends on its Dot/Icm type IV secretion system (T4SS), which delivers more than 300 effector proteins into the host, where they rewire cellular signaling to establish a replication-permissive niche, the Legionella-containing vacuole (LCV). Biogenesis of the LCV requires substantial redirection of vesicle trafficking and remodeling of intracellular membranes. In order to achieve this, several T4SS effectors target regulators of membrane trafficking, while others resemble lipases. Here, we characterized LpdA, a phospholipase D effector, which was previously proposed to modulate the lipid composition of the LCV. We found that ectopically expressed LpdA was targeted to the plasma membrane and Rab4- and Rab14-containing vesicles. Subcellular targeting of LpdA required a C-terminal motif, which is posttranslationally modified by S-palmitoylation. Substrate specificity assays showed that LpdA hydrolyzed phosphatidylinositol, -inositol-3- and -4-phosphate, and phosphatidylglycerol to phosphatidic acid (PA) in vitro. In HeLa cells, LpdA generated PA at vesicles and the plasma membrane. Imaging of different phosphatidylinositol phosphate (PIP) and organelle markers revealed that while LpdA did not impact on membrane association of various PIP probes, it triggered fragmentation of the Golgi apparatus. Importantly, although LpdA is translocated inefficiently into cultured cells, an L. pneumophila ΔlpdA mutant displayed reduced replication in murine lungs, suggesting that it is a virulence factor contributing to L. pneumophila infection in vivo.

  3. Phosphoproteomics of collagen receptor networks reveals SHP-2 phosphorylation downstream of wild-type DDR2 and its lung cancer mutants.

    PubMed

    Iwai, Leo K; Payne, Leo S; Luczynski, Maciej T; Chang, Francis; Xu, Huifang; Clinton, Ryan W; Paul, Angela; Esposito, Edward A; Gridley, Scott; Leitinger, Birgit; Naegle, Kristen M; Huang, Paul H

    2013-09-15

    Collagen is an important extracellular matrix component that directs many fundamental cellular processes including differentiation, proliferation and motility. The signalling networks driving these processes are propagated by collagen receptors such as the β1 integrins and the DDRs (discoidin domain receptors). To gain an insight into the molecular mechanisms of collagen receptor signalling, we have performed a quantitative analysis of the phosphorylation networks downstream of collagen activation of integrins and DDR2. Temporal analysis over seven time points identified 424 phosphorylated proteins. Distinct DDR2 tyrosine phosphorylation sites displayed unique temporal activation profiles in agreement with in vitro kinase data. Multiple clustering analysis of the phosphoproteomic data revealed several DDR2 candidate downstream signalling nodes, including SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2), NCK1 (non-catalytic region of tyrosine kinase adaptor protein 1), LYN, SHIP-2 [SH2 (Src homology 2)-domain-containing inositol phosphatase 2], PIK3C2A (phosphatidylinositol-4-phosphate 3-kinase, catalytic subunit type 2α) and PLCL2 (phospholipase C-like 2). Biochemical validation showed that SHP-2 tyrosine phosphorylation is dependent on DDR2 kinase activity. Targeted proteomic profiling of a panel of lung SCC (squamous cell carcinoma) DDR2 mutants demonstrated that SHP-2 is tyrosine-phosphorylated by the L63V and G505S mutants. In contrast, the I638F kinase domain mutant exhibited diminished DDR2 and SHP-2 tyrosine phosphorylation levels which have an inverse relationship with clonogenic potential. Taken together, the results of the present study indicate that SHP-2 is a key signalling node downstream of the DDR2 receptor which may have therapeutic implications in a subset of DDR2 mutations recently uncovered in genome-wide lung SCC sequencing screens.

  4. Function of AP2/ERF Transcription Factors Involved in the Regulation of Specialized Metabolism in Ophiorrhiza pumila Revealed by Transcriptomics and Metabolomics

    PubMed Central

    Udomsom, Nirin; Rai, Amit; Suzuki, Hideyuki; Okuyama, Jun; Imai, Ryosuke; Mori, Tetsuya; Nakabayashi, Ryo; Saito, Kazuki; Yamazaki, Mami

    2016-01-01

    The hairy roots (HR) of Ophiorrhiza pumila produce camptothecin (CPT), a monoterpenoid indole alkaloid used as a precursor in the synthesis of chemotherapeutic drugs. O. pumila HR culture is considered as a promising alternative source of CPT, however, the knowledge about the biosynthetic pathway and regulatory mechanism is still limited. In this study, five genes that encode AP2/ERF transcription factors, namely OpERF1–OpERF5, were isolated from HR of O. pumila. Phylogenetic analysis of AP2/ERF protein sequences suggested the close evolutionary relationship of OpERF1 with stress-responsive ERF factors in Arabidopsis and of OpERF2 with ERF factors reported to regulate alkaloid production, such as ORCA3 in Catharanthus roseus, NIC2 locus ERF in tobacco, and JRE4 in tomato. We generated the transgenic HR lines of O. pumila, ERF1i and ERF2i, in which the expression of OpERF1 and OpERF2, respectively, was suppressed using RNA interference technique. The transcriptome and metabolome of these suppressed HR were analyzed for functional characterization of OpERF1 and OpERF2. Although significant changes were not observed in the metabolome, including CPT and related compounds, the suppression of OpERF2 resulted in reduced expression of genes in the 2-C-methyl-d-erythritol 4-phosphate and secologanin-strictosidine pathways, which supply a precursor, strictosidine, for CPT biosynthesis. Furthermore, while it was not conclusive for OpERF1, enrichment analysis of differentially expressed genes in the suppressed HR showed that the gene ontology terms for oxidation-reduction, presumably involved in secondary metabolite pathways, were enriched in the ERF2i downregulated gene set. These results suggest a positive role of OpERF2 in regulating specialized metabolism in O. pumila. PMID:28018397

  5. Thermosensitive and mucoadhesive sol-gel composites of paclitaxel/dimethyl-β-cyclodextrin for buccal delivery.

    PubMed

    Choi, Soon Gil; Lee, Sang-Eun; Kang, Bong-Seok; Ng, Choon Lian; Davaa, Enkhzaya; Park, Jeong-Sook

    2014-01-01

    The purpose of this study was to develop a buccal paclitaxel delivery system using the thermosensitive polymer Pluronic F127 (PF127) and the mucoadhesive polymer polyethylene oxide (PEO). The anticancer agent paclitaxel is usually used to treat ovarian, breast, and non-small-cell lung cancer. To improve its aqueous solubility, paclitaxel was incorporated into an inclusion complex with (2,6-di-O-methyl)-β-cyclodextrin (DMβCD). The formation of the paclitaxel inclusion complex was evaluated using various techniques, including x-ray diffractometry (XRD), Fourier-transform infrared (FT-IR) spectrophotometry, differential scanning calorimetry (DSC), and scanning electron microscopy (SEM). Hydrogels were prepared using a cold method. Concentrations of 18, 20, and 23% (w/v) PF127 were dissolved in distilled water including paclitaxel and stored overnight in a refrigerator at 4 °C. PEO was added at concentrations of 0.1, 0.2, 0.4, 0.8, and 1% (w/v). Each formulation included paclitaxel (0.5 mg/mL). The sol-gel transition temperature of the hydrogels was measured using the tube-inverting method. Drug release from the hydrogels was measured using a Franz diffusion cell containing pH 7.4 phosphate-buffered solution (PBS) buffer at 37 °C. The cytotoxicity of each formulation was measured using the MTT assay with a human oral cancer cell (KB cell). The sol-gel transition temperature of the hydrogel decreased when PF127 was present and varied according to the presence of mucoadhesive polymers. The in vitro release was sustained and the release rate was slowed by the addition of the mucoadhesive polymer. The cytotoxicity of the blank formulation was low, although the drug-loaded hydrogel showed acceptable cytotoxicity. The results of our study suggest that the combination of a PF 127-based mucoadhesive hydrogel formulation and inclusion complexes improves the in vitro release and cytotoxic effect of paclitaxel.

  6. Phosphorus-Assisted Biomass Thermal Conversion: Reducing Carbon Loss and Improving Biochar Stability

    PubMed Central

    Zhao, Ling; Cao, Xinde; Zheng, Wei; Kan, Yue

    2014-01-01

    There is often over 50% carbon loss during the thermal conversion of biomass into biochar, leading to it controversy for the biochar formation as a carbon sequestration strategy. Sometimes the biochar also seems not to be stable enough due to physical, chemical, and biological reactions in soils. In this study, three phosphorus-bearing materials, H3PO4, phosphate rock tailing (PRT), and triple superphosphate (TSP), were used as additives to wheat straw with a ratio of 1: 0.4–0.8 for biochar production at 500°C, aiming to alleviate carbon loss during pyrolysis and to increase biochar-C stabilization. All these additives remarkably increased the biochar yield from 31.7% (unmodified biochar) to 46.9%–56.9% (modified biochars). Carbon loss during pyrolysis was reduced from 51.7% to 35.5%–47.7%. Thermogravimetric analysis curves showed that the additives had no effect on thermal stability of biochar but did enhance its oxidative stability. Microbial mineralization was obviously reduced in the modified biochar, especially in the TSP-BC, in which the total CO2 emission during 60-d incubation was reduced by 67.8%, compared to the unmodified biochar. Enhancement of carbon retention and biochar stability was probably due to the formation of meta-phosphate or C-O-PO3, which could either form a physical layer to hinder the contact of C with O2 and bacteria, or occupy the active sites of the C band. Our results indicate that pre-treating biomass with phosphors-bearing materials is effective for reducing carbon loss during pyrolysis and for increasing biochar stabilization, which provides a novel method by which biochar can be designed to improve the carbon sequestration capacity. PMID:25531111

  7. Characterization of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (HDS) gene from Ginkgo biloba.

    PubMed

    Kim, Sang-Min; Kim, Soo-Un

    2010-02-01

    Diterpene trilactone ginkgolides, one of the major constituents of Ginkgo biloba extract, have shown interesting bioactivities including platelet-activating factor antagonistic activity. 1-Hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (HDS), converting 2-C-methyl-d-erythritol-2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate, is the penultimate enzyme of the seven-step 2-C-methyl-d-erythritol 4-phosphate pathway that supplies building blocks for plant isoprenoids of plastid origin such as ginkgolides and carotenoids. Here, we report on the isolation and characterization of the full-length cDNA encoding HDS (GbHDS, GenBank accession number: DQ251630) from G. biloba. Full-length cDNA of GbHDS, 2,763 bp long, contained an ORF of 2,226 bp encoding a protein composed of 741 amino acids. The theoretical molecular weight and pI of the deduced mature GbHDS of 679 amino acid residues are 75.6 kDa and 5.5, respectively. From 2 weeks after initiation of the culture onward, transcription level of this gene in the ginkgo embryo roots increased to about two times higher than that in the leaves. GbHDS was predicted to possess chloroplast transit peptide of 62 amino acid residues, suggesting its putative localization in the plastids. The transient gene expression in Arabidopsis protoplasts confirmed that the transit peptide was capable of delivering the GbHDS protein from the cytosol into the chloroplasts. The isolation and characterization of GbHDS gene enabled us to further understand the role of GbHDS in the terpenoid biosynthesis in G. biloba.

  8. Metabolic engineering of Salmonella vaccine bacteria to boost human Vγ2Vδ2 T cell immunity.

    PubMed

    Workalemahu, Grefachew; Wang, Hong; Puan, Kia-Joo; Nada, Mohanad H; Kuzuyama, Tomohisa; Jones, Bradley D; Jin, Chenggang; Morita, Craig T

    2014-07-15

    Human Vγ2Vδ2 T cells monitor isoprenoid metabolism by recognizing foreign (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), a metabolite in the 2-C-methyl-D-erythritol-4-phosphate pathway used by most eubacteria and apicomplexan parasites, and self isopentenyl pyrophosphate, a metabolite in the mevalonate pathway used by humans. Whereas microbial infections elicit prolonged expansion of memory Vγ2Vδ2 T cells, immunization with prenyl pyrophosphates or aminobisphosphonates elicit short-term Vγ2Vδ2 expansion with rapid anergy and deletion upon subsequent immunizations. We hypothesized that a live, attenuated bacterial vaccine that overproduces HMBPP would elicit long-lasting Vγ2Vδ2 T cell immunity by mimicking a natural infection. Therefore, we metabolically engineered the avirulent aroA(-) Salmonella enterica serovar Typhimurium SL7207 strain by deleting the gene for LytB (the downstream enzyme from HMBPP) and functionally complementing for this loss with genes encoding mevalonate pathway enzymes. LytB(-) Salmonella SL7207 had high HMBPP levels, infected human cells as efficiently as did the wild-type bacteria, and stimulated large ex vivo expansions of Vγ2Vδ2 T cells from human donors. Importantly, vaccination of a rhesus monkey with live lytB(-) Salmonella SL7207 stimulated a prolonged expansion of Vγ2Vδ2 T cells without significant side effects or anergy induction. These studies provide proof-of-principle that metabolic engineering can be used to derive live bacterial vaccines that boost Vγ2Vδ2 T cell immunity. Similar engineering of metabolic pathways to produce lipid Ags or B vitamin metabolite Ags could be used to derive live bacterial vaccine for other unconventional T cells that recognize nonpeptide Ags.

  9. Riboflavin synthesis genes ribE, ribB, ribH, ribA reside in the lux operon of Photobacterium leiognathi.

    PubMed

    Lin, J W; Chao, Y F; Weng, S F

    2001-06-15

    Nucleotide sequence of the riboflavin synthesis genes ribE, ribB, ribH, ribA (GenBank Accession No. AF364106) resided in the lux operon of Photobacterium leiognathi PL741 has been determined, and the amino acid sequences of riboflavin synthetase (RibE), DHBP synthetase (RibB), lumazine synthetase (RibH), GTP cyclohydrolase II (RibA) encoded by the riboflavin synthesis genes are deduced. Nucleotide sequence reveals that the ribE gene encodes the riboflavin synthetase responsible for converting lumazine to riboflavin, the ribB gene encodes the DHBP synthetase responsible for 3,4-dihydroxyl-2-butanone 4-phosphate synthesis, the ribH gene encodes the lumazine synthetase responsible for lumazine synthesis, and the ribA gene encodes the GTP cyclohydrolase II responsible for lumazine synthesis. Functional analysis illustrates that the specific segments lay behind the ribH and ribA genes might form potential loops Omega(oT) and Omega(TI)--Omega(TII); Omega(oT) is functioned as mRNA stability loop or/and for subregulation by alternative modulation, and Omega(TI)--Omega(TII) could be the transcriptional terminator of the lux operon. The gene order of the ribE, ribB, ribH, ribA genes resided in the lux operon and linked to the lum operon is <--ter*-lumQ-lumP-R&R-luxC-luxD-luxA-luxB-luxN-luxE-luxG-ribE-ribB-ribH-ribA-ter--> (R&R: regulatory region; ter: transcriptional terminator), whereas the R&R is the regulatory region for the lum and the lux operons, and ter and ter* are the transcriptional terminators for the lux and lum operons.

  10. LuxR- and Acyl-Homoserine-Lactone-Controlled Non-lux Genes Define a Quorum-Sensing Regulon in Vibrio fischeri

    PubMed Central

    Callahan, Sean M.; Dunlap, Paul V.

    2000-01-01

    The luminescence (lux) operon (luxICDABEG) of the symbiotic bacterium Vibrio fischeri is regulated by the transcriptional activator LuxR and two acyl-homoserine lactone (acyl-HSL) autoinducers (the luxI-dependent 3-oxo-hexanoyl-HSL [3-oxo-C6-HSL] and the ainS-dependent octanoyl-HSL [C8-HSL]) in a population density-responsive manner called quorum sensing. To identify quorum-sensing-regulated (QSR) proteins different from those encoded by lux genes, we examined the protein patterns of V. fischeri quorum-sensing mutants defective in luxI, ainS, and luxR by two-dimensional polyacrylamide gel electrophoresis. Five non-Lux QSR proteins, QsrP, RibB, AcfA, QsrV, and QSR 7, were identified; their production occurred preferentially at high population density, required both LuxR and 3-oxo-C6-HSL, and was inhibited by C8-HSL at low population density. The genes encoding two of the QSR proteins were characterized: qsrP directs cells to synthesize an apparently novel periplasmic protein, and ribB is a homolog of the Escherichia coli gene for 3,4-dihydroxy-2-butanone 4-phosphate synthase, a key enzyme for riboflavin synthesis. The qsrP and ribB promoter regions each contained a sequence similar to the lux operon lux box, a 20-bp region of dyad symmetry necessary for LuxR/3-oxo-C6-HSL-dependent activation of lux operon transcription. V. fischeri qsrP and ribB mutants exhibited no distinct phenotype in culture. However, a qsrP mutant, in competition with its parent strain, was less successful in colonizing Euprymna scolopes, the symbiotic host of V. fischeri. The newly identified QSR genes, together with the lux operon, define a LuxR/acyl-HSL-responsive quorum-sensing regulon in V. fischeri. PMID:10781550

  11. Divergent Regulation of Terpenoid Metabolism in the Trichomes of Wild and Cultivated Tomato Species1[W][OA

    PubMed Central

    Besser, Katrin; Harper, Andrea; Welsby, Nicholas; Schauvinhold, Ines; Slocombe, Stephen; Li, Yi; Dixon, Richard A.; Broun, Pierre

    2009-01-01

    The diversification of chemical production in glandular trichomes is important in the development of resistance against pathogens and pests in two species of tomato. We have used genetic and genomic approaches to uncover some of the biochemical and molecular mechanisms that underlie the divergence in trichome metabolism between the wild species Solanum habrochaites LA1777 and its cultivated relative, Solanum lycopersicum. LA1777 produces high amounts of insecticidal sesquiterpene carboxylic acids (SCAs), whereas cultivated tomatoes lack SCAs and are more susceptible to pests. We show that trichomes of the two species have nearly opposite terpenoid profiles, consisting mainly of monoterpenes and low levels of sesquiterpenes in S. lycopersicum and mainly of SCAs and very low monoterpene levels in LA1777. The accumulation patterns of these terpenoids are different during development, in contrast to the developmental expression profiles of terpenoid pathway genes, which are similar in the two species, but they do not correlate in either case with terpenoid accumulation. However, our data suggest that the accumulation of monoterpenes in S. lycopersicum and major sesquiterpenes in LA1777 are linked both genetically and biochemically. Metabolite analyses after targeted gene silencing, inhibitor treatments, and precursor feeding all show that sesquiterpene biosynthesis relies mainly on products from the plastidic 2-C-methyl-d-erythritol-4-phosphate pathway in LA1777 but less so in the cultivated species. Furthermore, two classes of sesquiterpenes produced by the wild species may be synthesized from distinct pools of precursors via cytosolic and plastidial cyclases. However, highly trichome-expressed sesquiterpene cyclase-like enzymes were ruled out as being involved in the production of major LA1777 sesquiterpenes. PMID:18997116

  12. Alterations in Energy/Redox Metabolism Induced by Mitochondrial and Environmental Toxins: A Specific Role for Glucose-6-Phosphate-Dehydrogenase and the Pentose Phosphate Pathway in Paraquat Toxicity

    PubMed Central

    2015-01-01

    Parkinson’s disease (PD) is a multifactorial disorder with a complex etiology including genetic risk factors, environmental exposures, and aging. While energy failure and oxidative stress have largely been associated with the loss of dopaminergic cells in PD and the toxicity induced by mitochondrial/environmental toxins, very little is known regarding the alterations in energy metabolism associated with mitochondrial dysfunction and their causative role in cell death progression. In this study, we investigated the alterations in the energy/redox-metabolome in dopaminergic cells exposed to environmental/mitochondrial toxins (paraquat, rotenone, 1-methyl-4-phenylpyridinium [MPP+], and 6-hydroxydopamine [6-OHDA]) in order to identify common and/or different mechanisms of toxicity. A combined metabolomics approach using nuclear magnetic resonance (NMR) and direct-infusion electrospray ionization mass spectrometry (DI-ESI-MS) was used to identify unique metabolic profile changes in response to these neurotoxins. Paraquat exposure induced the most profound alterations in the pentose phosphate pathway (PPP) metabolome. 13C-glucose flux analysis corroborated that PPP metabolites such as glucose-6-phosphate, fructose-6-phosphate, glucono-1,5-lactone, and erythrose-4-phosphate were increased by paraquat treatment, which was paralleled by inhibition of glycolysis and the TCA cycle. Proteomic analysis also found an increase in the expression of glucose-6-phosphate dehydrogenase (G6PD), which supplies reducing equivalents by regenerating nicotinamide adenine dinucleotide phosphate (NADPH) levels. Overexpression of G6PD selectively increased paraquat toxicity, while its inhibition with 6-aminonicotinamide inhibited paraquat-induced oxidative stress and cell death. These results suggest that paraquat “hijacks” the PPP to increase NADPH reducing equivalents and stimulate paraquat redox cycling, oxidative stress, and cell death. Our study clearly demonstrates that alterations

  13. Engineering of Recombinant Poplar Deoxy-D-Xylulose-5-Phosphate Synthase (PtDXS) by Site-Directed Mutagenesis Improves Its Activity

    PubMed Central

    Banerjee, Aparajita; Preiser, Alyssa L.

    2016-01-01

    Deoxyxylulose 5-phosphate synthase (DXS), a thiamine diphosphate (ThDP) dependent enzyme, plays a regulatory role in the methylerythritol 4-phosphate (MEP) pathway. Isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), the end products of this pathway, inhibit DXS by competing with ThDP. Feedback inhibition of DXS by IDP and DMADP constitutes a significant metabolic regulation of this pathway. The aim of this work was to experimentally test the effect of key residues of recombinant poplar DXS (PtDXS) in binding both ThDP and IDP. This work also described the engineering of PtDXS to improve the enzymatic activity by reducing its inhibition by IDP and DMADP. We have designed and tested modifications of PtDXS in an attempt to reduce inhibition by IDP. This could possibly be valuable by removing a feedback that limits the usefulness of the MEP pathway in biotechnological applications. Both ThDP and IDP use similar interactions for binding at the active site of the enzyme, however, ThDP being a larger molecule has more anchoring sites at the active site of the enzyme as compared to the inhibitors. A predicted enzyme structure was examined to find ligand-enzyme interactions, which are relatively more important for inhibitor-enzyme binding than ThDP-enzyme binding, followed by their modifications so that the binding of the inhibitors can be selectively affected compared to ThDP. Two alanine residues important for binding ThDP and the inhibitors were mutated to glycine. In two of the cases, both the IDP inhibition and the overall activity were increased. In another case, both the IDP inhibition and the overall activity were reduced. This provides proof of concept that it is possible to reduce the feedback from IDP on DXS activity. PMID:27548482

  14. A carbamate-based approach to primaquine prodrugs: antimalarial activity, chemical stability and enzymatic activation.

    PubMed

    Mata, Graça; do Rosário, Virgílio E; Iley, Jim; Constantino, Luís; Moreira, Rui

    2012-01-15

    O-Alkyl and O-aryl carbamate derivatives of the antimalarial drug primaquine were synthesised as potential prodrugs that prevent oxidative deamination to the inactive metabolite carboxyprimaquine. Both O-alkyl and O-aryl carbamates undergo hydrolysis in alkaline and pH 7.4 phosphate buffers to the parent drug, with O-aryl carbamates being ca. 10(6)-10(10) more reactive than their O-alkyl counterparts. In human plasma O-alkyl carbamates were stable, whereas in contrast their O-aryl counterparts rapidly released the corresponding phenol product, with primaquine being released only slowly over longer incubation periods. Activation of the O-aryl carbamates in human plasma appears to be catalysed by butyrylcholinesterase (BuChE), which leads to carbamoylation of the catalytic serine of the enzyme followed by subsequent slow enzyme reactivation and release of parent drug. Most of the O-aryl and O-alkyl carbamates are activated in rat liver homogenates with half-lives ranging from 9 to 15 h, while the 4-nitrophenyl carbamate was hydrolysed too rapidly to determine an accurate rate constant. Antimalarial activity was studied using a model consisting of Plasmodium berghei, Balb C mice and Anopheles stephensi mosquitoes. When compared to controls, ethyl and n-hexyl carbamates were able to significantly reduce the percentage of infected mosquitos as well as the mean number of oocysts per infected mosquito, thus indicating that O-alkyl carbamates of primaquine have the potential to be developed as transmission-blocking antimalarial agents.

  15. Enhanced production of steviol glycosides in mycorrhizal plants: a concerted effect of arbuscular mycorrhizal symbiosis on transcription of biosynthetic genes.

    PubMed

    Mandal, Shantanu; Upadhyay, Shivangi; Singh, Ved Pal; Kapoor, Rupam

    2015-04-01

    Stevia rebaudiana (Bertoni) produces steviol glycosides (SGs)--stevioside (stev) and rebaudioside-A (reb-A) that are valued as low calorie sweeteners. Inoculation with arbuscular mycorrhizal fungi (AMF) augments SGs production, though the effect of this interaction on SGs biosynthesis has not been studied at molecular level. In this study transcription profiles of eleven key genes grouped under three stages of the SGs biosynthesis pathway were compared. The transcript analysis showed upregulation of genes encoding 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway enzymes viz.,1-deoxy-D-xylulose 5-phospate synthase (DXS), 1-deoxy-D-xylulose 5-phospate reductoisomerase (DXR) and 2-C-methyl-D-erytrithol 2,4-cyclodiphosphate synthase (MDS) in mycorrhizal (M) plants. Zn and Mn are imperative for the expression of MDS and their enhanced uptake in M plants could be responsible for the increased transcription of MDS. Furthermore, in the second stage of SGs biosynthesis pathway, mycorrhization enhanced the transcription of copalyl diphosphate synthase (CPPS) and kaurenoic acid hydroxylase (KAH). Their expression is decisive for SGs biosynthesis as CPPS regulates flow of metabolites towards synthesis of kaurenoid precursors and KAH directs these towards steviol synthesis instead of gibberellins. In the third stage glucosylation of steviol to reb-A by four specific uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) occurs. While higher transcription of all the three characterized UGTs in M plants explains augmented production of SGs; higher transcript levels of UGT76G1, specifically improved reb-A to stev ratio implying increased sweetness. The work signifies that AM symbiosis upregulates the transcription of all eleven SGs biosynthesis genes as a result of improved nutrition and enhanced sugar concentration due to increased photosynthesis in M plants.

  16. Linear Free Energy Relationship Analysis of Transition State Mimicry by 3-Deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) Oxime, a DAHP Synthase Inhibitor and Phosphate Mimic.

    PubMed

    Balachandran, Naresh; To, Frederick; Berti, Paul J

    2017-01-31

    3-Deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase catalyzes an aldol-like reaction of phosphoenolpyruvate (PEP) with erythrose 4-phosphate (E4P) to form DAHP in the first step of the shikimate biosynthetic pathway. DAHP oxime, in which an oxime replaces the ketone, is a potent inhibitor, with Ki = 1.5 μM. Linear free energy relationship (LFER) analysis of DAHP oxime inhibition using DAHP synthase mutants revealed an excellent correlation between transition state stabilization and inhibition. The equations of LFER analysis were rederived to formalize the possibility of proportional, rather than equal, changes in the free energies of transition state stabilization and inhibitor binding, in accord with the fact that the majority of LFER analyses in the literature demonstrate nonunity slopes. A slope of unity, m = 1, indicates that catalysis and inhibitor binding are equally sensitive to perturbations such as mutations or modified inhibitor/substrate structures. Slopes <1 or >1 indicate that inhibitor binding is less sensitive or more sensitive, respectively, to perturbations than is catalysis. LFER analysis using the tetramolecular specificity constant, that is, plotting log(KM,MnKM,PEPKM,E4P/kcat) versus log(Ki), revealed a slope, m, of 0.34, with r(2) = 0.93. This provides evidence that DAHP oxime is mimicking the first irreversible transition state of the DAHP synthase reaction, presumably phosphate departure from the tetrahedral intermediate. This is evidence that the oxime group can act as a functional, as well as structural, mimic of phosphate groups.

  17. Peripherally cross-linking the shell of core-shell polymer micelles decreases premature release of physically loaded combretastatin A4 in whole blood and increases its mean residence time and subsequent potency against primary murine breast tumors after IV administration

    PubMed Central

    Wakaskar, Rajesh R.; Bathena, Sai Praneeth R.; Tallapaka, Shailendra; Ambardekar, Vishakha V.; Gautum, Nagsen; Thakare, Rhishikesh N.; Simet, Samantha M.; Curran, Stephen M.; Singh, Rakesh K.; Dong, Yuxiang

    2014-01-01

    Purpose Determine the feasibility and potential benefit of peripherally cross-linking the shell of core-shell polymer micelles on the premature release of physically loaded hydrophobic drug in whole blood and subsequent potency against solid tumors. Methods Individual Pluronic F127 polymer micelles (F127 PM) peripherally cross-linked with ethylenediamine at 76% of total PEO blocks (X-F127 PM) were physically loaded with combretastatin A4 (CA4) by the solid dispersion method and compared to CA4 physically loaded in uncross-linked F127 PM, CA4 in DMSO in vitro, or water-soluble CA4 phosphate (CA4P) in vivo. Results X-F127 PM had similar CA4 loading and aqueous solubility as F127 PM up to 10 mg CA4 / mL at 22.9 wt% and did not aggregate in PBS or 90% (v/v) human serum at 37°C for at least 24 h. In contrast, X-F127 PM decreased the unbound fraction of CA4 in whole blood (fu) and increased the mean plasma residence time and subsequent potency of CA4 against the vascular function and growth of primary murine 4T1 breast tumors over CA4 in F127 PM and water-soluble CA4P after IV administration. Conclusions Given that decreasing the fu is an indication of decreased drug release, peripherally cross-linking the shell of core-shell polymer micelles may be a simple approach to decrease premature release of physically loaded hydrophobic drug in the blood and increase subsequent potency in solid tumors. PMID:25223962

  18. Prerequisite for highly efficient isoprenoid production by cyanobacteria discovered through the over-expression of 1-deoxy-d-xylulose 5-phosphate synthase and carbon allocation analysis.

    PubMed

    Kudoh, Kai; Kawano, Yusuke; Hotta, Shingo; Sekine, Midori; Watanabe, Takafumi; Ihara, Masaki

    2014-07-01

    Cyanobacteria have recently been receiving considerable attention owing to their potential as photosynthetic producers of biofuels and biomaterials. Here, we focused on the production of isoprenoids by cyanobacteria, and aimed to provide insight into metabolic engineering design. To this end, we examined the over-expression of a key enzyme in 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, 1-deoxy-d-xylulose 5-phosphate synthase (DXS) in the cyanobacterium Synechocystis sp. PCC6803. In the DXS-over-expression strain (Dxs_ox), the mRNA and protein levels of DXS were 4-times and 1.5-times the levels in the wild-type (WT) strain, respectively. The carotenoid content of the Dxs_ox strain (8.4 mg/g dry cell weight [DCW]) was also up to 1.5-times higher than that in the WT strain (5.6 mg/g DCW), whereas the glycogen content dramatically decreased to an undetectable level. These observations suggested that the carotenoid content in the Dxs_ox strain was increased by consuming glycogen, which is a C-storage compound in cyanobacteria. We also quantified the total sugar (145 and 104 mg/g DCW), total fatty acids (31 and 24 mg/g DCW) and total protein (200 and 240 mg/g DCW) content in the WT and Dxs_ox strains, respectively, which were much higher than the carotenoid content. In particular, approximately 54% of the proteins were phycobiliproteins. This study demonstrated the major destinations of carbon flux in cyanobacteria, and provided important insights into metabolic engineering. Target yield can be improved through optimization of gene expression, the DXS protein stabilization, cell propagation depression and restriction of storage compound synthesis.

  19. Modulation of phosphoinositide metabolism in aortic smooth muscle cells by allylamine

    SciTech Connect

    Cox, L.R.; Murphy, S.K.; Ramos, K. )

    1990-08-01

    Aortic smooth muscle cells (SMC) modulate from a contractile to a proliferative phenotype upon subchronic exposure to allylamine. The present studies were designed to determine if this phenotypic modulation is associated with alterations in the metabolism of membrane phosphoinositides. 32P incorporation into phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2), and phosphatidic acid (PA) was lower by 31, 35, and 22%, respectively, in SMC from allylamine-treated animals relative to controls. In contrast, incorporation of (3H)myoinositol into inositol phosphates did not differ in allylamine cells relative to control cells. Exposure to dibutyryl (db) cAMP (0.2 mM) and theophylline (0.1 mM) reduced 32P incorporation into PIP and PIP2 in SMC from both experimental groups. Under these conditions, a decrease in (3H)myoinositol incorporation into inositol 1-phosphate was only observed in allylamine cells. The effects of db cAMP and theophylline in allylamine and control SMC correlated with a marked decrease in cellular proliferation. These results suggest that alterations in phosphoinositide synthesis and/or degradation contribute to the enhanced proliferation of SMC induced by allylamine. To further examine this concept, the effects of agents which modulate protein kinase C (PKC) activity were evaluated. Sphingosine (125-500 ng/ml), a PKC inhibitor, decreased SMC proliferation in allylamine, but not control cells. 12-O-Tetradecanoylphorbol-13-acetate (1-100 ng/ml), a PKC agonist, stimulated proliferation in control cells, but inhibited proliferation in cells from allylamine-treated animals. We conclude that allylamine-induced phenotypic modulation of SMC is associated with alterations in phosphoinositide metabolism.

  20. Comparative Transcriptomics Unravel Biochemical Specialization of Leaf Tissues of Stevia for Diterpenoid Production1

    PubMed Central

    Kim, Mi Jung; Jin, Jingjing; Zheng, Junshi

    2015-01-01

    Stevia (Stevia rebaudiana) produces not only a group of diterpenoid glycosides known as steviol glycosides (SGs), but also other labdane-type diterpenoids that may be spatially separated from SGs. However, their biosynthetic routes and spatial distribution in leaf tissues have not yet been elucidated. Here, we integrate metabolome and transcriptome analyses of Stevia to explore the biosynthetic capacity of leaf tissues for diterpenoid metabolism. Tissue-specific chemical analyses confirmed that SGs were accumulated in leaf cells but not in trichomes. On the other hand, Stevia leaf trichomes stored other labdane-type diterpenoids such as oxomanoyl oxide and agatholic acid. RNA sequencing analyses from two different tissues of Stevia provided a comprehensive overview of dynamic metabolic activities in trichomes and leaf without trichomes. These metabolite-guided transcriptomics and phylogenetic and gene expression analyses clearly identified specific gene members encoding enzymes involved in the 2-C-methyl-d-erythritol 4-phosphate pathway and the biosynthesis of steviol or other labdane-type diterpenoids. Additionally, our RNA sequencing analysis uncovered copalyl diphosphate synthase (SrCPS) and kaurene synthase1 (SrKS1) homologs, SrCPS2 and KS-like (SrKSL), which were specifically expressed in trichomes. In vitro and in planta assays showed that unlike SrCPS and SrKS1, SrCPS2 synthesized labda-13-en-8-ol diphosphate and successively catalyzed the formation of manoyl oxide and epi-manoyl oxide in combination with SrKSL. Our findings suggest that Stevia may have evolved to use distinct metabolic pathways to avoid metabolic interferences in leaf tissues for efficient production of diverse secondary metabolites. PMID:26438788

  1. Platelet-activating factor stimulates metabolism of phosphoinositides via phospholipase A2 in primary cultured rat hepatocytes

    SciTech Connect

    Okayasu, T.; Hasegawa, K.; Ishibashi, T.

    1987-07-01

    Addition of platelet-activating factor (PAF) to cells doubly labeled with (/sup 14/C)glycerol plus (/sup 3/H)arachidonic acid resulted in a transient decrease of (/sup 14/C)glycerol-labeled phosphatidylinositol (PI) and a transient increase of (/sup 14/C)glycerol-labeled lysophosphatidylinositol (LPI). (/sup 3/H)Arachidonate-labeled PI, on the other hand, decreased in a time-dependent manner. The radioactivity in phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, and phosphatidylserine did not change significantly. The /sup 3/H//sup 14/C ratio decreased in PI in a time-dependent manner, suggesting the involvement of a phospholipase A2 activity. Although PAF also induced a gradual increase of diacylglycerol (DG), the increase of (/sup 14/C)glycerol-labeled DG paralleled the loss of triacyl (/sup 14/C)glycerol and the /sup 3/H//sup 14/C ratio of DG was 16 times smaller than that of PI. Thus, DG seemed not to be derived from PI. In myo- (/sup 3/H)inositol-prelabeled cells, PAF induced a transient decrease of (/sup 3/H)phosphatidylinositol-4,5-bis-phosphate (TPI) and (/sup 3/H)phosphatidylinositol-4-phosphate (DPI) at 1 min. PAF stimulation of cultured hepatocytes prelabeled with /sup 32/Pi induced a transient decrease of (/sup 32/P)polyphosphoinositides at 20 sec to 1 min. (/sup 32/P)LPI appeared within 10 sec after stimulation and paralleled the loss of (/sup 32/P)PI. (/sup 3/H)Inositol triphosphate, (/sup 3/H)inositol diphosphate, and (/sup 3/H)inositol phosphate, which increased in a time-dependent manner upon stimulation with adrenaline, did not accumulate with the stimulation due to PAF. These observations indicate that PAF causes degradation of inositol phospholipids via phospholipase A2 and induces a subsequent resynthesis of these phospholipids.

  2. Phosphatidylinositol 4-kinase IIβ negatively regulates invadopodia formation and suppresses an invasive cellular phenotype

    PubMed Central

    Alli-Balogun, Ganiyu Olabanji; Gewinner, Christina A.; Jacobs, Ruth; Kriston-Vizi, Janos; Waugh, Mark G.; Minogue, Shane

    2016-01-01

    The type II phosphatidylinositol 4-kinase (PI4KII) enzymes synthesize the lipid phosphatidylinositol 4-phosphate (PI(4)P), which has been detected at the Golgi complex and endosomal compartments and recruits clathrin adaptors. Despite common mechanistic similarities between the isoforms, the extent of their redundancy is unclear. We found that depletion of PI4KIIα and PI4KIIβ using small interfering RNA led to actin remodeling. Depletion of PI4KIIβ also induced the formation of invadopodia containing membrane type I matrix metalloproteinase (MT1-MMP). Depletion of PI4KII isoforms also differentially affected trans-Golgi network (TGN) pools of PI(4)P and post-TGN traffic. PI4KIIβ depletion caused increased MT1-MMP trafficking to invasive structures at the plasma membrane and was accompanied by reduced colocalization of MT1-MMP with membranes containing the endosomal markers Rab5 and Rab7 but increased localization with the exocytic Rab8. Depletion of PI4KIIβ was sufficient to confer an aggressive invasive phenotype on minimally invasive HeLa and MCF-7 cell lines. Mining oncogenomic databases revealed that loss of the PI4K2B allele and underexpression of PI4KIIβ mRNA are associated with human cancers. This finding supports the cell data and suggests that PI4KIIβ may be a clinically significant suppressor of invasion. We propose that PI4KIIβ synthesizes a pool of PI(4)P that maintains MT1-MMP traffic in the degradative pathway and suppresses the formation of invadopodia. PMID:27798239

  3. The casein kinases Yck1p and Yck2p act in the secretory pathway, in part, by regulating the Rab exchange factor Sec2p

    PubMed Central

    Stalder, Danièle; Novick, Peter J.

    2016-01-01

    Sec2p is a guanine nucleotide exchange factor that activates Sec4p, the final Rab GTPase of the yeast secretory pathway. Sec2p is recruited to secretory vesicles by the upstream Rab Ypt32p acting in concert with phosphatidylinositol-4-phosphate (PI(4)P). Sec2p also binds to the Sec4p effector Sec15p, yet Ypt32p and Sec15p compete against each other for binding to Sec2p. We report here that the redundant casein kinases Yck1p and Yck2p phosphorylate sites within the Ypt32p/Sec15p binding region and in doing so promote binding to Sec15p and inhibit binding to Ypt32p. We show that Yck2p binds to the autoinhibitory domain of Sec2p, adjacent to the PI(4)P binding site, and that addition of PI(4)P inhibits Sec2p phosphorylation by Yck2p. Loss of Yck1p and Yck2p function leads to accumulation of an intracellular pool of the secreted glucanase Bgl2p, as well as to accumulation of Golgi-related structures in the cytoplasm. We propose that Sec2p is phosphorylated after it has been recruited to secretory vesicles and the level of PI(4)P has been reduced. This promotes Sec2p function by stimulating its interaction with Sec15p. Finally, Sec2p is dephosphorylated very late in the exocytic reaction to facilitate recycling. PMID:26700316

  4. Comparative Transcriptomics Unravel Biochemical Specialization of Leaf Tissues of Stevia for Diterpenoid Production.

    PubMed

    Kim, Mi Jung; Jin, Jingjing; Zheng, Junshi; Wong, Limsoon; Chua, Nam-Hai; Jang, In-Cheol

    2015-12-01

    Stevia (Stevia rebaudiana) produces not only a group of diterpenoid glycosides known as steviol glycosides (SGs), but also other labdane-type diterpenoids that may be spatially separated from SGs. However, their biosynthetic routes and spatial distribution in leaf tissues have not yet been elucidated. Here, we integrate metabolome and transcriptome analyses of Stevia to explore the biosynthetic capacity of leaf tissues for diterpenoid metabolism. Tissue-specific chemical analyses confirmed that SGs were accumulated in leaf cells but not in trichomes. On the other hand, Stevia leaf trichomes stored other labdane-type diterpenoids such as oxomanoyl oxide and agatholic acid. RNA sequencing analyses from two different tissues of Stevia provided a comprehensive overview of dynamic metabolic activities in trichomes and leaf without trichomes. These metabolite-guided transcriptomics and phylogenetic and gene expression analyses clearly identified specific gene members encoding enzymes involved in the 2-C-methyl-d-erythritol 4-phosphate pathway and the biosynthesis of steviol or other labdane-type diterpenoids. Additionally, our RNA sequencing analysis uncovered copalyl diphosphate synthase (SrCPS) and kaurene synthase1 (SrKS1) homologs, SrCPS2 and KS-like (SrKSL), which were specifically expressed in trichomes. In vitro and in planta assays showed that unlike SrCPS and SrKS1, SrCPS2 synthesized labda-13-en-8-ol diphosphate and successively catalyzed the formation of manoyl oxide and epi-manoyl oxide in combination with SrKSL. Our findings suggest that Stevia may have evolved to use distinct metabolic pathways to avoid metabolic interferences in leaf tissues for efficient production of diverse secondary metabolites.

  5. Overexpression of SrUGT85C2 from Stevia reduced growth and yield of transgenic Arabidopsis by influencing plastidial MEP pathway.

    PubMed

    Guleria, Praveen; Masand, Shikha; Yadav, Sudesh Kumar

    2014-04-15

    The transcript expression of a gene SrUGT85C2 has been documented for direct relation with steviol glycoside content in Stevia plant. Steviol glycoside and gibberellin biosynthetic routes are divergent branches of methyl erythritol-4 phosphate (MEP) pathway. So, SrUGT85C2 might be an influencing gibberellin content. Hence in the present study, transgenic Arabidopsis thaliana overexpressing SrUGT85C2 cDNA from Stevia rebaudiana was developed to check its effect on gibberellin accumulation and related plant growth parameters. The developed transgenics showed a noteworthy decrease of 78-83% in GA3 content. Moreover, the transgenics showed a gibberellin deficient phenotype comprising stunted hypocotyl length, reduced shoot growth and a significant fall in relative water content. Transgenics also showed 17-37 and 64-76% reduction in chlorophyll a and chlorophyll b contents, respectively. Reduction in photosynthetic pigments could be responsible for the noticed significant decrease in plant biomass. Like steviol glycoside and gibberellin biosynthesis, chlorophyll biosynthesis also occurs from the precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) of MEP pathway in the plastids. The observed downregulated expression of genes encoding MEP pathway enzymes geranyl geranyl diphosphate synthase (GGDPS), copalyl diphosphate synthase (CDPS), kaurenoic acid oxidase (KAO), chlorophyll synthetase and chlorophyll a oxygenase in transgenics overexpressing SrUGT85C2 might be responsible for the reduction in gibberellins as well as chlorophyll. This study has documented for the first time the regulatory role of SrUGT85C2 in the biosynthesis of steviol glycoside, gibberellins and chlorophyll.

  6. Dose-dependent elimination of 8-methoxypsoralen in the mouse

    SciTech Connect

    Cheney, P.; Pacula, C.M.; Gerber, N.; Mays, D.C.

    1986-03-01

    8-Methoxypsoralen (8-MOP), a photoactive linear furocoumarin, is effective in the treatment of several diseases, including psoriasis, mycosis fungoides and T-cell leukemia. Recently, a specific extraction procedure for /sup 14/C-8-MOP showed that the elimination of 8-MOP in the rat was dose-dependent. Similar pharmacokinetic studies were undertaken in mice. Purity of /sup 14/C-8-MOP, verified by a four-tube countercurrent distribution using hexane (8 ml) and pH 7.4 phosphate buffer (0.1 M 15 ml) as described by Bush, was >98% and distributed with a partition coefficient of 3.86. Male CD-1 mice were each given an i.p. dose of 10 or 50 mg/kg of /sup 14/C-8-MOP (3.4 ..mu..Ci/mg) sacrificed at timed intervals, homogenized in 150 ml of 0.1 M phosphate buffer (pH 7.4) and a portion (0.8 ml) of the homogenate used to quantify 8-MOP as described above. The elimination half-life measured in the first 45 min was 7.4 min at 10 mg/kg and 95 min at 50 mg/kg. A similar half-life of 9.2 min was measured in mice given an i.v. dose 10 mg/kg of 8-MOP. Explanations of dose-dependent elimination include enzyme saturation, product inhibition or both. Between 58-80% of the administered radioactivity was recovered in the urine within 24 hr. Nine peaks of radioactivity were observed in the urine by HPLC, two of which coeluted with 5,8-dihydroxypsoralen and 6-(7-hydroxy-8-methoxycoumaryl)-acetic acid.

  7. Metal-catalyzed oxidation of phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli: inactivation and destabilization by oxidation of active-site cysteines.

    PubMed

    Park, O K; Bauerle, R

    1999-03-01

    The in vitro instability of the phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase [DAHPS(Phe)] from Escherichia coli has been found to be due to a metal-catalyzed oxidation mechanism. DAHPS(Phe) is one of three differentially feedback-regulated isoforms of the enzyme which catalyzes the first step of aromatic biosynthesis, the formation of DAHP from phosphoenolpyruvate and D-erythrose-4-phosphate. The activity of the apoenzyme decayed exponentially, with a half-life of about 1 day at room temperature, and the heterotetramer slowly dissociated to the monomeric state. The enzyme was stabilized by the presence of phosphoenolpyruvate or EDTA, indicating that in the absence of substrate, a trace metal(s) was the inactivating agent. Cu2+ and Fe2+, but none of the other divalent metals that activate the enzyme, greatly accelerated the rate of inactivation and subunit dissociation. Both anaerobiosis and the addition of catalase significantly reduced Cu2+-catalyzed inactivation. In the spontaneously inactivated enzyme, there was a net loss of two of the seven thiols per subunit; this value increased with increasing concentrations of added Cu2+. Dithiothreitol completely restored the enzymatic activity and the two lost thiols in the spontaneously inactivated enzyme but was only partially effective in reactivation of the Cu2+-inactivated enzyme. Mutant enzymes with conservative replacements at either of the two active-site cysteines, Cys61 or Cys328, were insensitive to the metal attack. Peptide mapping of the Cu2+-inactivated enzyme revealed a disulfide linkage between these two cysteine residues. All results indicate that DAHPS(Phe) is a metal-catalyzed oxidation system wherein bound substrate protects active-site residues from oxidative attack catalyzed by bound redox metal cofactor. A mechanism of inactivation of DAHPS is proposed that features a metal redox cycle that requires the sequential oxidation of its two active-site cysteines.

  8. Biosynthesis of sesquiterpenes in grape berry exocarp of Vitis vinifera L.: evidence for a transport of farnesyl diphosphate precursors from plastids to the cytosol.

    PubMed

    May, Bianca; Lange, B Markus; Wüst, Matthias

    2013-11-01

    The participation of the mevalonic acid (MVA) and 1-deoxy-d-xylulose 5-phosphate/2-C-methyl-d-erythritol-4-phosphate (DOXP/MEP) pathways in sesquiterpene biosynthesis of grape berries was investigated. There is an increasing interest in this class of terpenoids, since the oxygenated sesquiterpene rotundone was identified as the peppery aroma impact compound in Australian Shiraz wines. To investigate precursor supply pathway utilization, in vivo feeding experiments were performed with the deuterium labeled, pathway specific, precursors [5,5-(2)H2]-1-deoxy-d-xylulose and [5,5-(2)H2]-mevalonic acid lactone. Head Space-Solid Phase Micro Extraction-Gas Chromatography-Mass Spectrometry (HS-SPME-GC-MS) analysis of the generated volatile metabolites demonstrated that de novo sesquiterpene biosynthesis is mainly located in the grape berry exocarp (skin), with no detectable activity in the mesocarp (flesh) of the Lemberger variety. Interestingly, precursors from both the (primarily) cytosolic MVA and plastidial DOXP/MEP pathways were incorporated into grape sesquiterpenes in the varieties Lemberger, Gewürztraminer and Syrah. Our labeling data provide evidence for a homogenous, cytosolic pool of precursors for sesquiterpene biosynthesis, indicating that a transport of precursors occurs mostly from plastids to the cytosol. The labeling patterns of the sesquiterpene germacrene D were in agreement with a cyclization mechanism analogous to that of a previously cloned enantioselective (R)-germacrene D synthase from Solidago canadensis. This observation was subsequently confirmed by enantioselective GC-MS analysis demonstrating the exclusive presence of (R)-germacrene D, and not the (S)-enantiomer, in grape berries.

  9. Cyclic nucleotide regulation of inositol lipid metabolism in rat cerebral cortex

    SciTech Connect

    Nicchitta, C.V.; Williamson, J.R.

    1986-05-01

    The authors have investigated the effects of compounds known to elevate cAMP levels and/or activate the cAMP dependent protein kinase on resting and stimulated inositol lipid metabolism in rat cortical slices and synaptosomes. In (/sup 3/H)-myo-inositol-labeled brain slices, carbamylcholine-stimulated accumulation of (/sup 3/H)-myo-inositol 1-phosphate (IP/sub 1/) was decreased 50% by forskolin (150..mu..M) isobutylmethylxantihine (IBMX) (1mM), 8-bromo cAMP (5mM) or 8-(4-chlorophenylthio) cAMP (5mM). In studies with synaptosomes, the incorporation of (/sup 32/Pi) into phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP/sub 2/) and phosphatidic acid (PA) was also reduced in the presence of forskolin or the cAMP phosphodiesterase inhibitors IBMX and RO20-1724 (+/-)-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone. In the presence of maximally effective concentrations of these agents, steady state PIP levels were reduced by 50% and PIP/sub 2/ and PA levels by 40%. The effects of these compounds on (/sup 32/Pi) labeling of the phosphoinositides and PA are consistent with an inhibition of flux through the inositide cycle, presumably through the action of cAMP. Such an inhibition may account for the reduction in carbamylcholine sensitive IP/sub 1/ accumulation observed in brain slices. These studies indicate that in rat brain, ..beta..-adrenergic stimulation may be involved in modulating the activity of muscarinic cholinergic agonists. This regulation appears to be at the level of inositol lipid metabolism.

  10. Phosphate tungsten bronze series: crystallographic and structural properties of low-dimensional conductors.

    PubMed

    Roussel, P; Pérez, O; Labbé, P

    2001-10-01

    Phosphate tungsten bronzes have been shown to be conductors of low dimensionality. A review of the crystallographic and structural properties of this huge series of compounds is given here, corresponding to the present knowledge of the different X-ray studies and electron microscopy investigations. Three main families are described, monophosphate tungsten bronzes, Ax(PO2)4(WO3)2m, either with pentagonal tunnels (MPTBp) or with hexagonal tunnels (MPTBh), and diphosphate tungsten bronzes, Ax(P2O4)2(WO3)2m, mainly with hexagonal tunnels (DPTBh). The general aspect of these crystal structures may be described as a building of polyhedra sharing oxygen corners made of regular stacking of WO3-type slabs with a thickness function of m, joined by slices of tetrahedral PO4 phosphate or P2O7 diphosphate groups. The relations of the different slabs with respect to the basic perovskite structure are mentioned. The structural description is focused on the tilt phenomenon of the WO6 octahedra inside a slab of WO3-type. In this respect, a comparison with the different phases of the WO3 crystal structures is established. The various modes of tilting and the different possible connections between two adjacent WO3-type slabs involve a great variety of structures with different symmetries, as well as the existence of numerous twins in MPTBp's. Several phase transitions, with the appearance of diffuse scattering and modulation phenomena, were analysed by X-ray scattering measurements and through the temperature dependence of various physical properties for the MPTBp's. The role of the W displacements within the WO3-type slabs, in two modulated structures (m = 4 and m = 10), already solved, is discussed. Finally, the complexity of the structural aspects of DPTBh's is explained on the basis of the average structures which are the only ones solved.

  11. De novo assembly and transcriptome analysis of the rubber tree (Hevea brasiliensis) and SNP markers development for rubber biosynthesis pathways.

    PubMed

    Mantello, Camila Campos; Cardoso-Silva, Claudio Benicio; da Silva, Carla Cristina; de Souza, Livia Moura; Scaloppi Junior, Erivaldo José; de Souza Gonçalves, Paulo; Vicentini, Renato; de Souza, Anete Pereira

    2014-01-01

    Hevea brasiliensis (Willd. Ex Adr. Juss.) Muell.-Arg. is the primary source of natural rubber that is native to the Amazon rainforest. The singular properties of natural rubber make it superior to and competitive with synthetic rubber for use in several applications. Here, we performed RNA sequencing (RNA-seq) of H. brasiliensis bark on the Illumina GAIIx platform, which generated 179,326,804 raw reads on the Illumina GAIIx platform. A total of 50,384 contigs that were over 400 bp in size were obtained and subjected to further analyses. A similarity search against the non-redundant (nr) protein database returned 32,018 (63%) positive BLASTx hits. The transcriptome analysis was annotated using the clusters of orthologous groups (COG), gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Pfam databases. A search for putative molecular marker was performed to identify simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs). In total, 17,927 SSRs and 404,114 SNPs were detected. Finally, we selected sequences that were identified as belonging to the mevalonate (MVA) and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways, which are involved in rubber biosynthesis, to validate the SNP markers. A total of 78 SNPs were validated in 36 genotypes of H. brasiliensis. This new dataset represents a powerful information source for rubber tree bark genes and will be an important tool for the development of microsatellites and SNP markers for use in future genetic analyses such as genetic linkage mapping, quantitative trait loci identification, investigations of linkage disequilibrium and marker-assisted selection.

  12. OSBP-Related Protein Family: Mediators of Lipid Transport and Signaling at Membrane Contact Sites.

    PubMed

    Kentala, Henriikka; Weber-Boyvat, Marion; Olkkonen, Vesa M

    2016-01-01

    Oxysterol-binding protein (OSBP) and its related protein homologs, ORPs, constitute a conserved family of lipid-binding/transfer proteins (LTPs) expressed ubiquitously in eukaryotes. The ligand-binding domain of ORPs accommodates cholesterol and oxysterols, but also glycerophospholipids, particularly phosphatidylinositol-4-phosphate (PI4P). ORPs have been implicated as intracellular lipid sensors or transporters. Most ORPs carry targeting determinants for the endoplasmic reticulum (ER) and non-ER organelle membrane. ORPs are located and function at membrane contact sites (MCSs), at which ER is closely apposed with other organelle limiting membranes. Such sites have roles in lipid transport and metabolism, control of Ca(2+) fluxes, and signaling events. ORPs are postulated either to transport lipids over MCSs to maintain the distinct lipid compositions of organelle membranes, or to control the activity of enzymes/protein complexes with functions in signaling and lipid metabolism. ORPs may transfer PI4P and another lipid class bidirectionally. Transport of PI4P followed by its hydrolysis would in this model provide the energy for transfer of the other lipid against its concentration gradient. Control of organelle lipid compositions by OSBP/ORPs is important for the life cycles of several pathogenic viruses. Targeting ORPs with small-molecular antagonists is proposed as a new strategy to combat viral infections. Several ORPs are reported to modulate vesicle transport along the secretory or endocytic pathways. Moreover, antagonists of certain ORPs inhibit cancer cell proliferation. Thus, ORPs are LTPs, which mediate interorganelle lipid transport and coordinate lipid signals with a variety of cellular regimes.

  13. Alterations in energy/redox metabolism induced by mitochondrial and environmental toxins: a specific role for glucose-6-phosphate-dehydrogenase and the pentose phosphate pathway in paraquat toxicity.

    PubMed

    Lei, Shulei; Zavala-Flores, Laura; Garcia-Garcia, Aracely; Nandakumar, Renu; Huang, Yuting; Madayiputhiya, Nandakumar; Stanton, Robert C; Dodds, Eric D; Powers, Robert; Franco, Rodrigo

    2014-09-19

    Parkinson's disease (PD) is a multifactorial disorder with a complex etiology including genetic risk factors, environmental exposures, and aging. While energy failure and oxidative stress have largely been associated with the loss of dopaminergic cells in PD and the toxicity induced by mitochondrial/environmental toxins, very little is known regarding the alterations in energy metabolism associated with mitochondrial dysfunction and their causative role in cell death progression. In this study, we investigated the alterations in the energy/redox-metabolome in dopaminergic cells exposed to environmental/mitochondrial toxins (paraquat, rotenone, 1-methyl-4-phenylpyridinium [MPP+], and 6-hydroxydopamine [6-OHDA]) in order to identify common and/or different mechanisms of toxicity. A combined metabolomics approach using nuclear magnetic resonance (NMR) and direct-infusion electrospray ionization mass spectrometry (DI-ESI-MS) was used to identify unique metabolic profile changes in response to these neurotoxins. Paraquat exposure induced the most profound alterations in the pentose phosphate pathway (PPP) metabolome. 13C-glucose flux analysis corroborated that PPP metabolites such as glucose-6-phosphate, fructose-6-phosphate, glucono-1,5-lactone, and erythrose-4-phosphate were increased by paraquat treatment, which was paralleled by inhibition of glycolysis and the TCA cycle. Proteomic analysis also found an increase in the expression of glucose-6-phosphate dehydrogenase (G6PD), which supplies reducing equivalents by regenerating nicotinamide adenine dinucleotide phosphate (NADPH) levels. Overexpression of G6PD selectively increased paraquat toxicity, while its inhibition with 6-aminonicotinamide inhibited paraquat-induced oxidative stress and cell death. These results suggest that paraquat "hijacks" the PPP to increase NADPH reducing equivalents and stimulate paraquat redox cycling, oxidative stress, and cell death. Our study clearly demonstrates that alterations in

  14. Encapsulating drugs in biodegradable ultrafine fibers through co-axial electrospinning.

    PubMed

    Huang, Zheng-Ming; He, Chuang-Long; Yang, Aizhao; Zhang, Yanzhong; Han, Xiao-Jian; Yin, Junlin; Wu, Qingsheng

    2006-04-01

    This article describes an electrospinning process to fabricate double-layered ultrafine fibers. A bioabsorbable polymer, Polycaprolactone (PCL), was used as the outer layer or the shell and two medically pure drugs, Resveratrol (RT, a kind of antioxidant) and Gentamycin Sulfate (GS, an antibiotic), were used as the inner layers or the cores. Morphology and microstructure of the ultrafine fibers were characterized by scanning electron microscope (SEM) and transmission electron microscopy (TEM), whereas mechanical performance of them was understood through tensile test. In vitro degradation rates of the nanofibrous membranes were determined by measuring their weight loss when immersed in pH 7.4 phosphate-buffered saline (PBS) mixed with certain amount of Pseudomonas lipase for a maximum of 7 days. The drug release behaviors of the RT and GS were measured using a high performance liquid chromatography (HPLC) and ultraviolet-visible (UV-vis) spectroscopy, respectively. It has been found that the drug solutions without any fiber-forming additive could be encapsulated in the PCL ultrafine fibers, although they alone cannot be made into a fiber form. Beads on the fiber surface influenced the tensile behavior of the ultrafine fibers remarkably. When the core solvent was miscible with the shell solvent, higher drug concentration decreased the bead formation and thus favored the mechanical performance. The situation, however, became different if the two solvents were immiscible with each other. The degradation rate was closely related to hydrophilicity of the drugs in the cores. Higher hydrophilicity apparently led to faster degradation. The release profiles of the RT and GS exhibited a sustained release characteristic, with no burst release phenomenon.

  15. Crystal structure of 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE) from Mycobacterium tuberculosis.

    PubMed

    Shan, Shan; Chen, Xuehui; Liu, Ting; Zhao, Hanchao; Rao, Zihe; Lou, Zhiyong

    2011-05-01

    Isoprenoid precursors, which are a large group of natural products and play key roles in many biological pathways, can only be biosynthesized by the 2-C-methyl-d-erythritol 4-phosphate pathway in Mycobacterium tuberculosis. The 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase (IspE), which is an essential enzyme in the isoprenoid precursor biosynthesis pathway, catalyzes ATP-dependent phosphorylation of 4-diphosphocytidyl-2-C-methyl-d-erythritol (CDP-ME) to 4-diphosphocytidyl-2C-methyl-d-erythritol-2-phosphate and plays a crucial role in M. tuberculosis survival. Therefore, IspE is characterized as an attractive and potential target for antimicrobial drug discovery. However, no experimental structure of M. tuberculosis IspE has been reported, which has hindered our understanding of its structural details and mechanism of action. Here, we report the expression and purification of fully active full-length M. tuberculosis IspE and solve the high-resolution crystal structures of IspE alone and in complex with either the substrate CDP-ME or nonhydrolyzable ATP analog or ADP. The structures present a characteristic galactose/homoserine/mevalonate/phosphomevalonate kinase superfamily α/β-fold with a catalytic center located in a cleft between 2 domains and display clear substrate and ATP binding pockets. Our results also indicate distinct differences in ligand binding of M. tuberculosis IspE with other reported IspEs. Combined with the results of mutagenesis and enzymatic studies, our results provide useful information on the structural basis of IspE for future anti-M. tuberculosis drug discovery targeting this kinase.

  16. Changes in phosphoinositide turnover, Ca sup 2+ mobilization, and protein phosphorylation in platelets from NIDDM patients

    SciTech Connect

    Ishii, H.; Umeda, F.; Hashimoto, T.; Nawata, H. )

    1990-12-01

    Enhanced platelet functions have been demonstrated in patients with non-insulin-dependent diabetes mellitus (NIDDM). This study evaluated abnormalities in platelet signal transduction in diabetic patients, including turnover of phosphoinositides, mobilization of intracellular Ca2+, and phosphorylation of 20,000- and 47,000-Mr proteins (P20 and P47). Washed platelets were obtained from 6 patients with NIDDM whose platelet aggregation rates were abnormally elevated (DM-A group), 11 NIDDM patients with normal platelet aggregation rates (DM-B group), and 8 age-matched healthy control subjects. The mass and specific radioactivity of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol (PI), and phosphatidic acid (PA) in 32P-labeled platelets were not different among the three groups. Hydrolysis of PIP2, PIP, and PI; accumulation of PA; and phosphorylation of P20 in platelets stimulated by 0.05 U/ml thrombin were significantly increased in the DM-A group compared with the control or DM-B group. There was no difference in P47 phosphorylation among the three groups. On the contrary, P20 and P47 phosphorylation induced by 50 nM of 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C, was significantly decreased in the DM-A group. Additionally, the intracellular free Ca2+ concentration (( Ca2+)i) was measured with the fluorescent Ca2+ indicator fura 2. Although the basal (Ca2+)i value was similar in the three groups, the rise in (Ca2+)i induced by 0.05 U/ml thrombin in the presence and the absence of extracellular Ca2+ was significantly higher in the DM-A group than the other groups.

  17. SN-38-cyclodextrin complexation and its influence on the solubility, stability, and in vitro anticancer activity against ovarian cancer.

    PubMed

    Vangara, Kiran Kumar; Ali, Hamed Ismail; Lu, Dai; Liu, Jingbo Louise; Kolluru, Srikanth; Palakurthi, Srinath

    2014-04-01

    SN-38, an active metabolite of irinotecan, is up to 1,000-fold more potent than irinotecan. But the clinical use of SN-38 is limited by its extreme hydrophobicity and instability at physiological pH. To enhance solubility and stability, SN-38 was complexed with different cyclodextrins (CDs), namely, sodium sulfobutylether β-cyclodextrin (SBEβCD), hydroxypropyl β-cyclodextrin, randomly methylated β-cyclodextrin, and methyl β-cyclodextrin, and their influence on SN-38 solubility, stability, and in vitro cytotoxicity was studied against ovarian cancer cell lines (A2780 and 2008). Phase solubility studies were conducted to understand the pattern of SN-38 solubilization. SN-38-βCD complexes were characterized by differential scanning calorimetry (DSC), X-ray powder diffraction analysis (XRPD), and Fourier transform infrared (FTIR). Stability of SN-38-SBEβCD complex in pH 7.4 phosphate-buffered saline was evaluated and compared against free SN-38. Phase solubility studies revealed that SN-38 solubility increased linearly as a function of CD concentration and the linearity was characteristic of an AP-type system. Aqueous solubility of SN-38 was enhanced by about 30-1,400 times by CD complexation. DSC, XRPD, and FTIR studies confirmed the formation of inclusion complexes, and stability studies revealed that cyclodextrin complexation significantly increased the hydrolytic stability of SN-38 at physiological pH 7.4. Cytotoxicity of SN-38-SBEβCD complex was significantly higher than SN-38 and irinotecan in both A2780 and 2008 cell lines. Results suggest that SBEβCD encapsulated SN-38 deep into the cavity forming stable inclusion complex and as a result increased the solubility, stability, and cytotoxicity of SN-38. It may be concluded that preparation of inclusion complexes with SBEβCD is a suitable approach to overcome the solubility and stability problems of SN-38 for future clinical applications.

  18. Novel insights into structure–function mechanism and tissue-specific expression profiling of full-length dxr gene from Cymbopogon winterianus

    PubMed Central

    Devi, Kamalakshi; Dehury, Budheswar; Phukon, Munmi; Modi, Mahendra Kumar; Sen, Priyabrata

    2015-01-01

    The 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR; EC1.1.1.267), an NADPH-dependent reductase, plays a pivotal role in the methylerythritol 4-phosphate pathway (MEP), in the conversion of 1-deoxy-d-xylulose-5-phosphate (DXP) into MEP. The sheath and leaf of citronella (Cymbopogon winterianus) accumulates large amount of terpenes and sesquiterpenes with proven medicinal value and economic uses. Thus, sequencing of full length dxr gene and its characterization seems to be a valuable resource in metabolic engineering to alter the flux of isoprenoid active ingredients in plants. In this study, full length DXR from citronella was characterized through in silico and tissue-specific expression studies to explain its structure–function mechanism, mode of cofactor recognition and differential expression. The modelled DXR has a three-domain architecture and its active site comprised of a cofactor (NADPH) binding pocket and the substrate-binding pocket. Molecular dynamics simulation studies indicated that DXR model retained most of its secondary structure during 10 ns simulation in aqueous solution. The modelled DXR superimposes well with its closest structural homolog but subtle variations in the charge distribution over the cofactor recognition site were noticed. Molecular docking study revealed critical residues aiding tight anchoring NADPH within the active pocket of DXR. Tissue-specific differential expression analysis using semi-quantitative RT-PCR and qRT-PCR in various tissues of citronella plant revealed distinct differential expression of DXR. To our knowledge, this is the first ever report on DXR from the important medicinal plant citronella and further characterization of this gene will open up better avenues for metabolic engineering of secondary metabolite pathway genes from medicinal plants in the near future. PMID:25941629

  19. Bisphosphonate Inhibitors Reveal a Large Elasticity of Plastidic Isoprenoid Synthesis Pathway in Isoprene-Emitting Hybrid Aspen1

    PubMed Central

    2015-01-01

    Recently, a feedback inhibition of the chloroplastic 1-deoxy-d-xylulose 5-phosphate (DXP)/2-C-methyl-d-erythritol 4-phosphate (MEP) pathway of isoprenoid synthesis by end products dimethylallyl diphosphate (DMADP) and isopentenyl diphosphate (IDP) was postulated, but the extent to which DMADP and IDP can build up is not known. We used bisphosphonate inhibitors, alendronate and zoledronate, that inhibit the consumption of DMADP and IDP by prenyltransferases to gain insight into the extent of end product accumulation and possible feedback inhibition in isoprene-emitting hybrid aspen (Populus tremula × Populus tremuloides). A kinetic method based on dark release of isoprene emission at the expense of substrate pools accumulated in light was used to estimate the in vivo pool sizes of DMADP and upstream metabolites. Feeding with fosmidomycin, an inhibitor of DXP reductoisomerase, alone or in combination with bisphosphonates was used to inhibit carbon input into DXP/MEP pathway or both input and output. We observed a major increase in pathway intermediates, 3- to 4-fold, upstream of DMADP in bisphosphonate-inhibited leaves, but the DMADP pool was enhanced much less, 1.3- to 1.5-fold. In combined fosmidomycin/bisphosphonate treatment, pathway intermediates accumulated, reflecting cytosolic flux of intermediates that can be important under strong metabolic pull in physiological conditions. The data suggested that metabolites accumulated upstream of DMADP consist of phosphorylated intermediates and IDP. Slow conversion of the huge pools of intermediates to DMADP was limited by reductive energy supply. These data indicate that the DXP/MEP pathway is extremely elastic, and the presence of a significant pool of phosphorylated intermediates provides an important valve for fine tuning the pathway flux. PMID:25926480

  20. Pheophytinase Knockdown Impacts Carbon Metabolism and Nutraceutical Content Under Normal Growth Conditions in Tomato.

    PubMed

    Lira, Bruno Silvestre; Rosado, Daniele; Almeida, Juliana; de Souza, Amanda Pereira; Buckeridge, Marcos Silveira; Purgatto, Eduardo; Guyer, Luzia; Hörtensteiner, Stefan; Freschi, Luciano; Rossi, Magdalena

    2016-03-01

    Although chlorophyll (Chl) degradation is an essential biochemical pathway for plant physiology, our knowledge regarding this process still has unfilled gaps. Pheophytinase (PPH) was shown to be essential for Chl breakdown in dark-induced senescent leaves. However, the catalyzing enzymes involved in pigment turnover and fruit ripening-associated degreening are still controversial. Chl metabolism is closely linked to the biosynthesis of other isoprenoid-derived compounds, such as carotenoids and tocopherols, which are also components of the photosynthetic machinery. Chls, carotenoids and tocopherols share a common precursor, geranylgeranyl diphosphate, produced by the plastidial methylerythritol 4-phosphate (MEP) pathway. Additionally, the Chl degradation-derived phytol can be incorporated into tocopherol biosynthesis. In this context, tomato turns out to be an interesting model to address isoprenoid-metabolic cross-talk since fruit ripening combines degreening and an intensely active MEP leading to carotenoid accumulation. Here, we investigate the impact of PPH deficiency beyond senescence by the comprehensive phenotyping of SlPPH-knockdown tomato plants. In leaves, photosynthetic parameters indicate altered energy usage of excited Chl. As a mitigatory effect, photosynthesis-associated carotenoids increased while tocopherol content remained constant. Additionally, starch and soluble sugar profiles revealed a distinct pattern of carbon allocation in leaves that suggests enhanced sucrose exportation. The higher levels of carbohydrates in sink organs down-regulated carotenoid biosynthesis. Additionally, the reduction in Chl-derived phytol recycling resulted in decreased tocopherol content in transgenic ripe fruits. Summing up, tocopherol and carotenoid metabolism, together with the antioxidant capacity of the hydrophilic and hydrophobic fractions, were differentially affected in leaves and fruits of the transgenic plants. Thus, in tomato, PPH plays a role beyond

  1. Seedling growth responses to soil resources in the understory of a wet tropical forest.

    PubMed

    Holste, Ellen K; Kobe, Richard K; Vriesendorp, Corine F

    2011-09-01

    Plant growth responses to resources may be an important mechanism that influences species' distributions, coexistence, and community structure. Irradiance is considered the most important resource for seedling growth in the understory of wet tropical forests, but multiple soil nutrients and species have yet to be examined simultaneously with irradiance under field conditions. To identify potentially limiting resources, we modeled tree seedling growth as a function of irradiance and soil nutrients across five sites, spanning a soil fertility gradient in old-growth, wet tropical forests at La Selva Biological Station, Costa Rica. We measured an array of soil nutrients including total nitrogen (total N), inorganic N (nitrate [NO3-] and ammonium [NH4+]), phosphate (PO4-), and sum of base cations (SBC; potassium, magnesium, and calcium). Shade in the forest understory did not preclude seedling growth correlations with soil nutrients. Irradiance was a significant predictor of growth in 52% of the species, inorganic N in 54% (NO3- in 32%; NH4+ in 34%), total N in 47%, SBC in 39%, and PO4- in 29%. Overall, growth was correlated with both irradiance and soil nutrients in 45% of species and with soil nutrients only in an additional 48%; rarely was irradiance alone correlated with growth. Contrary to expectations, the magnitudes of growth effects, assessed as the maximum growth response to significant resources for each species, were similar for irradiance and most soil nutrients. Among species whose growth correlated with soil nutrients, the rank importance of nutrient effects was SBC, followed by N (total N, NO3-, and/or NH4+) and PO4-. Species' growth responsiveness (i.e., magnitudes of effect) to irradiance and soil nutrients was negatively correlated with species' shade tolerance (survival under 1% full sun). In this broad survey of species and resources, the nearly ubiquitous effects of soil nutrients on seedling growth challenge the idea that soil nutrients are less

  2. Trapping effect on a small molecular drug with vascular-disrupting agent CA4P in rodent H22 hepatic tumor model: in vivo magnetic resonance imaging and postmortem inductively coupled plasma atomic emission spectroscopy.

    PubMed

    Gao, Meng; Yao, Nan; Huang, Dejian; Jiang, Cuihua; Feng, Yuanbo; Li, Yue; Lou, Bin; Peng, Fei; Sun, Ziping; Ni, Yicheng; Zhang, Jian

    2015-06-01

    The aim of the present study is to verify the trapping effect of combretastatin A-4-phosphate (CA4P) on small molecular drugs in rodent tumors. Mice with H22 hepatocarcinoma were randomized into groups A and B. Magnetic resonance imaging (MRI) of T1WI, T2WI, and DWI was performed as baseline. Mice in group A were injected with Gd-DTPA and PBS. Mice in group B were injected with Gd-DTPA and CA4P. All mice undergo CE-T1WI at 0 h, 3 h, 6 h, 12 h, and 24 h. Enhancing efficacy of the two groups on CE-T1WI was compared with the signal-to-noise ratio (SNR) calculated. Concentrations of gadolinium measured by ICP-AES in the tumor were compared between groups. On the early CE-T1WI, tumors were equally enhanced in both groups. On the delayed CE-T1WI, the enhancing effect of group A was weaker than that of group B. The SNR and the concentration of gadolinium within the tumor of group A were lower than that of group B at 6 h, 12 h, and 24 h after administration. This study indicates that CA4P could improve the retention of Gd-DTPA in the tumor and MRI allowed dynamically monitoring trapping effects of CA4P on local retention of Gd-DTPA as a small molecular drug.

  3. Indirect Stimulation of Human Vγ2Vδ2 T cells Through Alterations in Isoprenoid Metabolism1

    PubMed Central

    Wang, Hong; Sarikonda, Ghanashyam; Puan, Kia-Joo; Tanaka, Yoshimasa; Feng, Ju; Giner, José-Luis; Cao, Rong; Mönkkönen, Jukka; Oldfield, Eric; Morita, Craig T.

    2011-01-01

    Human Vγ2Vδ2 T cells monitor isoprenoid metabolism by recognizing (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), an intermediate in the 2-C-methyl-D-erythritol-4-phosphate pathway used by microbes, and isopentenyl pyrophosphate (IPP), an intermediate in the mevalonate pathway used by humans. Aminobisphosphonates and alkylamines indirectly stimulate Vγ2Vδ2 cells by inhibiting farnesyl diphosphate synthase (FDPS) in the mevalonate pathway, thereby increasing IPP/ApppI that directly stimulate. In this study, we further characterize stimulation by these compounds, and define pathways used by new classes of compounds. Consistent with FDPS inhibition, stimulation of Vγ2Vδ2 cells by aminobisphosphonates and alkylamines was much more sensitive to statin inhibition than stimulation by prenyl pyrophosphates. However, the continuous presence of aminobisphosphonates was toxic for T cells, and blocked their proliferation. Aminobisphosphonate stimulation was rapid and prolonged, independent of known antigen presenting molecules, and resistant to fixation. New classes of stimulatory compounds–mevalonate, the alcohol of HMBPP, and alkenyl phosphonates–likely stimulate differently. Mevalonate, a rate-limiting metabolite, appears to enter cells to increase IPP levels whereas the alcohol of HMBPP and alkenyl phosphonates are directly recognized. The critical chemical feature of bisphosphonates is the amino moiety, because its loss switched aminobisphosphonates to direct antigens. Transfection of APC with siRNA downregulating FDPS rendered them stimulatory for Vγ2Vδ2 cells, and increased cellular IPP. siRNAs for isopentenyl diphosphate isomerase functioned similarly. Our results show that a variety of manipulations affecting isoprenoid metabolism lead to stimulation of Vγ2Vδ2 T cells and that pulsing aminobisphosphonates would be more effective for the ex vivo expansion of Vγ2Vδ2 T cells for adoptive cancer immunotherapy. PMID:22013129

  4. Drug-in-cyclodextrin-in-liposomes: A novel drug delivery system for flurbiprofen.

    PubMed

    Zhang, Lina; Zhang, Qi; Wang, Xin; Zhang, Wenji; Lin, Congcong; Chen, Fen; Yang, Xinggang; Pan, Weisan

    2015-08-15

    A novel delivery system based on drug-cyclodextrin (CD) complexation and liposomes has been developed to improve therapeutic effect. Three different means, i.e., co-evaporation (COE), co-ground (GR) and co-lyophilization (COL) and three different CDs (β-CD, HP-β-CD and SBE-β-CD) were contrasted to investigate the characteristics of the end products. FP/FP-CD loaded liposomes were obtained by thin layer evaporation technique. Size, zeta potential and encapsulation efficiency were investigated by light scattering analysis and minicolumn centrifugation. Differential scanning calorimetry (DSC) and transmission electron microscopy (TEM) showed the amorphous form of complexes and spherical morphology of FP-HP-β-CD COE loaded liposomes. The pH 7.4 phosphate buffer solution (PBS) was selected as the medium for the in vitro release. Wistar rats were put into use to study the pharmacokinetic behavior in vivo. FP-HP-β-CD COE loaded liposomes showed the better physicochemical characters that followed the average particle size, polydispersity index, zeta potential and mean encapsulation efficiency 158±10 nm, 0.19±0.1, -12.4±0.1 mW and 56.1±0.5%, separately. The relative bioavailability of FP-HP-β-CD COE loaded liposomes was 420%, 201% and 402% compared with FP solution, FP-HP-β-CD and FP-liposomes, respectively. In conclusion, the novel delivery system improved the relative bioavailability of FP significantly and provided a perspective way for delivery of insoluble drugs.

  5. Dynamics of Phosphoinositide-Dependent Signaling in Sympathetic Neurons

    PubMed Central

    Kruse, Martin; Vivas, Oscar; Traynor-Kaplan, Alexis

    2016-01-01

    In neurons, loss of plasma membrane phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] leads to a decrease in exocytosis and changes in electrical excitability. Restoration of PI(4,5)P2 levels after phospholipase C activation is therefore essential for a return to basal neuronal activity. However, the dynamics of phosphoinositide metabolism have not been analyzed in neurons. We measured dynamic changes of PI(4,5)P2, phosphatidylinositol 4-phosphate, diacylglycerol, inositol 1,4,5-trisphosphate, and Ca2+ upon muscarinic stimulation in sympathetic neurons from adult male Sprague-Dawley rats with electrophysiological and optical approaches. We used this kinetic information to develop a quantitative description of neuronal phosphoinositide metabolism. The measurements and analysis show and explain faster synthesis of PI(4,5)P2 in sympathetic neurons than in electrically nonexcitable tsA201 cells. They can be used to understand dynamic effects of receptor-mediated phospholipase C activation on excitability and other PI(4,5)P2-dependent processes in neurons. SIGNIFICANCE STATEMENT Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is a minor phospholipid in the cytoplasmic leaflet of the plasma membrane. Depletion of PI(4,5)P2 via phospholipase C-mediated hydrolysis leads to a decrease in exocytosis and alters electrical excitability in neurons. Restoration of PI(4,5)P2 is essential for a return to basal neuronal activity. However, the dynamics of phosphoinositide metabolism have not been analyzed in neurons. We studied the dynamics of phosphoinositide metabolism in sympathetic neurons upon muscarinic stimulation and used the kinetic information to develop a quantitative description of neuronal phosphoinositide metabolism. The measurements and analysis show a several-fold faster synthesis of PI(4,5)P2 in sympathetic neurons than in an electrically nonexcitable cell line, and provide a framework for future studies of PI(4,5)P2-dependent processes in neurons. PMID:26818524

  6. 3-hydroxybenzene 1,2,4-trisphosphate, a novel second messenger mimic and unusual substrate for type-I myo-inositol 1,4,5-trisphosphate 5-phosphatase: Synthesis and physicochemistry.

    PubMed

    Mills, Stephen J; Dozol, Hélène; Vandeput, Fabrice; Backers, Katrien; Woodman, Timothy; Erneux, Christophe; Spiess, Bernard; Potter, Barry V L

    2006-11-01

    3-Hydroxybenzene 1,2,4-trisphosphate 4 is a new myo-inositol 1,4,5-trisphosphate analogue based on the core structure of benzene 1,2,4-trisphosphate 2 with an additional hydroxyl group at position-3, and is the first noninositol based compound to be a substrate for inositol 1,4,5-trisphosphate 5-phosphatase. In physicochemical studies on 2, when three equivalents of protons were added, the (31)P NMR spectrum displayed monophasic behaviour in which phosphate-1 and phosphate-2 behaved independently in most of the studied pH range. For compound 4, phosphate-2 and phosphate-4 interacted with the 3-OH group, which does not titrate at physiological pH, displaying complex biphasic behaviour which demonstrated co-operativity between these groups. Phosphate-1 and phosphate-2 strongly interacted with each other and phosphate-4 experienced repulsion because of the interaction of the 3-OH group. Benzene 1,2,4-trisphosphate 2 is resistant to inositol 1,4,5-trisphosphate type I 5-phosphatase catalysed dephosphorylation. However, surprisingly, 3-hydroxybenzene 1,2,4-trisphosphate 4 was dephosphorylated by this 5-phosphatase to give the symmetrical 2,3-dihydroxybenzene 1,4-bisphosphate 16. The extra hydroxyl group is shown to form a hydrogen bond with the vicinal phosphate groups at -15 degrees C, and (1)H NMR titration of the ring and hydroxyl protons in 4 shows the OH proton to be strongly stabilized as soon as the phosphate groups are deprotonated. The effect of the phenolic 3-OH group in compound 4 confirms a critical role for the 6-OH group of the natural messenger in the dephosphorylation mechanism that persists even in radically modified analogues.

  7. Transcriptome Sequencing and Expression Analysis of Terpenoid Biosynthesis Genes in Litsea cubeba

    PubMed Central

    Han, Xiao-Jiao; Wang, Yang-Dong; Chen, Yi-Cun; Lin, Li-Yuan; Wu, Qing-Ke

    2013-01-01

    Background Aromatic essential oils extracted from fresh fruits of Litsea cubeba (Lour.) Pers., have diverse medical and economic values. The dominant components in these essential oils are monoterpenes and sesquiterpenes. Understanding the molecular mechanisms of terpenoid biosynthesis is essential for improving the yield and quality of terpenes. However, the 40 available L. cubeba nucleotide sequences in the public databases are insufficient for studying the molecular mechanisms. Thus, high-throughput transcriptome sequencing of L. cubeba is necessary to generate large quantities of transcript sequences for the purpose of gene discovery, especially terpenoid biosynthesis related genes. Results Using Illumina paired-end sequencing, approximately 23.5 million high-quality reads were generated. De novo assembly yielded 68,648 unigenes with an average length of 834 bp. A total of 38,439 (56%) unigenes were annotated for their functions, and 35,732 and 25,806 unigenes could be aligned to the GO and COG database, respectively. By searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG), 16,130 unigenes were assigned to 297 KEGG pathways, and 61 unigenes, which contained the mevalonate and 2-C-methyl-D-erythritol 4-phosphate pathways, could be related to terpenoid backbone biosynthesis. Of the 12,963 unigenes, 285 were annotated to the terpenoid pathways using the PlantCyc database. Additionally, 14 terpene synthase genes were identified from the transcriptome. The expression patterns of the 16 genes related to terpenoid biosynthesis were analyzed by RT-qPCR to explore their putative functions. Conclusion RNA sequencing was effective in identifying a large quantity of sequence information. To our knowledge, this study is the first exploration of the L. cubeba transcriptome, and the substantial amount of transcripts obtained will accelerate the understanding of the molecular mechanisms of essential oils biosynthesis. The results may help

  8. Bisphosphonate inhibitors reveal a large elasticity of plastidic isoprenoid synthesis pathway in isoprene-emitting hybrid aspen.

    PubMed

    Rasulov, Bahtijor; Talts, Eero; Kännaste, Astrid; Niinemets, Ülo

    2015-06-01

    Recently, a feedback inhibition of the chloroplastic 1-deoxy-D-xylulose 5-phosphate (DXP)/2-C-methyl-D-erythritol 4-phosphate (MEP) pathway of isoprenoid synthesis by end products dimethylallyl diphosphate (DMADP) and isopentenyl diphosphate (IDP) was postulated, but the extent to which DMADP and IDP can build up is not known. We used bisphosphonate inhibitors, alendronate and zoledronate, that inhibit the consumption of DMADP and IDP by prenyltransferases to gain insight into the extent of end product accumulation and possible feedback inhibition in isoprene-emitting hybrid aspen (Populus tremula × Populus tremuloides). A kinetic method based on dark release of isoprene emission at the expense of substrate pools accumulated in light was used to estimate the in vivo pool sizes of DMADP and upstream metabolites. Feeding with fosmidomycin, an inhibitor of DXP reductoisomerase, alone or in combination with bisphosphonates was used to inhibit carbon input into DXP/MEP pathway or both input and output. We observed a major increase in pathway intermediates, 3- to 4-fold, upstream of DMADP in bisphosphonate-inhibited leaves, but the DMADP pool was enhanced much less, 1.3- to 1.5-fold. In combined fosmidomycin/bisphosphonate treatment, pathway intermediates accumulated, reflecting cytosolic flux of intermediates that can be important under strong metabolic pull in physiological conditions. The data suggested that metabolites accumulated upstream of DMADP consist of phosphorylated intermediates and IDP. Slow conversion of the huge pools of intermediates to DMADP was limited by reductive energy supply. These data indicate that the DXP/MEP pathway is extremely elastic, and the presence of a significant pool of phosphorylated intermediates provides an important valve for fine tuning the pathway flux.

  9. Engineering of Recombinant Poplar Deoxy-D-Xylulose-5-Phosphate Synthase (PtDXS) by Site-Directed Mutagenesis Improves Its Activity.

    PubMed

    Banerjee, Aparajita; Preiser, Alyssa L; Sharkey, Thomas D

    2016-01-01

    Deoxyxylulose 5-phosphate synthase (DXS), a thiamine diphosphate (ThDP) dependent enzyme, plays a regulatory role in the methylerythritol 4-phosphate (MEP) pathway. Isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), the end products of this pathway, inhibit DXS by competing with ThDP. Feedback inhibition of DXS by IDP and DMADP constitutes a significant metabolic regulation of this pathway. The aim of this work was to experimentally test the effect of key residues of recombinant poplar DXS (PtDXS) in binding both ThDP and IDP. This work also described the engineering of PtDXS to improve the enzymatic activity by reducing its inhibition by IDP and DMADP. We have designed and tested modifications of PtDXS in an attempt to reduce inhibition by IDP. This could possibly be valuable by removing a feedback that limits the usefulness of the MEP pathway in biotechnological applications. Both ThDP and IDP use similar interactions for binding at the active site of the enzyme, however, ThDP being a larger molecule has more anchoring sites at the active site of the enzyme as compared to the inhibitors. A predicted enzyme structure was examined to find ligand-enzyme interactions, which are relatively more important for inhibitor-enzyme binding than ThDP-enzyme binding, followed by their modifications so that the binding of the inhibitors can be selectively affected compared to ThDP. Two alanine residues important for binding ThDP and the inhibitors were mutated to glycine. In two of the cases, both the IDP inhibition and the overall activity were increased. In another case, both the IDP inhibition and the overall activity were reduced. This provides proof of concept that it is possible to reduce the feedback from IDP on DXS activity.

  10. The role of phosphate in a multistep enzymatic reaction: reactions of the substrate and intermediate in pieces.

    PubMed

    Kholodar, Svetlana A; Allen, C Leigh; Gulick, Andrew M; Murkin, Andrew S

    2015-02-25

    Several mechanistically unrelated enzymes utilize the binding energy of their substrate's nonreacting phosphoryl group to accelerate catalysis. Evidence for the involvement of the phosphodianion in transition state formation has come from reactions of the substrate in pieces, in which reaction of a truncated substrate lacking its phosphorylmethyl group is activated by inorganic phosphite. What has remained unknown until now is how the phosphodianion group influences the reaction energetics at different points along the reaction coordinate. 1-Deoxy-D-xylulose-5-phosphate (DXP) reductoisomerase (DXR), which catalyzes the isomerization of DXP to 2-C-methyl-D-erythrose 4-phosphate (MEsP) and subsequent NADPH-dependent reduction, presents a unique opportunity to address this concern. Previously, we have reported the effect of covalently linked phosphate on the energetics of DXP turnover. Through the use of chemically synthesized MEsP and its phosphate-truncated analogue, 2-C-methyl-D-glyceraldehyde, the current study revealed a loss of 6.1 kcal/mol of kinetic barrier stabilization upon truncation, of which 4.4 kcal/mol was regained in the presence of phosphite dianion. The activating effect of phosphite was accompanied by apparent tightening of its interactions within the active site at the intermediate stage of the reaction, suggesting a role of the phosphodianion in disfavoring intermediate release and in modulation of the on-enzyme isomerization equilibrium. The results of kinetic isotope effect and structural studies indicate rate limitation by physical steps when the covalent linkage is severed. These striking differences in the energetics of the natural reaction and the reactions in pieces provide a deeper insight into the contribution of enzyme-phosphodianion interactions to the reaction coordinate.

  11. Assignment of function to a domain of unknown function: DUF1537 is a new kinase family in catabolic pathways for acid sugars

    PubMed Central

    Zhang, Xinshuai; Carter, Michael S.; Vetting, Matthew W.; San Francisco, Brian; Zhao, Suwen; Al-Obaidi, Nawar F.; Solbiati, Jose O.; Thiaville, Jennifer J.; de Crécy-Lagard, Valérie; Jacobson, Matthew P.; Almo, Steven C.; Gerlt, John A.

    2016-01-01

    Using a large-scale “genomic enzymology” approach, we (i) assigned novel ATP-dependent four-carbon acid sugar kinase functions to members of the DUF1537 protein family (domain of unknown function; Pfam families PF07005 and PF17042) and (ii) discovered novel catabolic pathways for d-threonate, l-threonate, and d-erythronate. The experimentally determined ligand specificities of several solute binding proteins (SBPs) for TRAP (tripartite ATP-independent permease) transporters for four-carbon acids, including d-erythronate and l-erythronate, were used to constrain the substrates for the catabolic pathways that degrade the SBP ligands to intermediates in central carbon metabolism. Sequence similarity networks and genome neighborhood networks were used to identify the enzyme components of the pathways. Conserved genome neighborhoods encoded SBPs as well as permease components of the TRAP transporters, members of the DUF1537 family, and a member of the 4-hydroxy-l-threonine 4-phosphate dehydrogenase (PdxA) oxidative decarboxylase, class II aldolase, or ribulose 1,5-bisphosphate carboxylase/oxygenase, large subunit (RuBisCO) superfamily. Because the characterized substrates of members of the PdxA, class II aldolase, and RuBisCO superfamilies are phosphorylated, we postulated that the members of the DUF1537 family are novel ATP-dependent kinases that participate in catabolic pathways for four-carbon acid sugars. We determined that (i) the DUF1537/PdxA pair participates in a pathway for the conversion of d-threonate to dihydroxyacetone phosphate and CO2 and (ii) the DUF1537/class II aldolase pair participates in pathways for the conversion of d-erythronate and l-threonate (epimers at carbon-3) to dihydroxyacetone phosphate and CO2. The physiological importance of these pathways was demonstrated in vivo by phenotypic and genetic analyses. PMID:27402745

  12. Rare earth elements in the phosphatic-enriched sediment of the Peru shelf

    USGS Publications Warehouse

    Piper, D.Z.; Baedecker, P.A.; Crock, J.G.; Burnett, W.C.; Loebner, B.J.

    1988-01-01

    Apatite-enriched materials from the Peru shelf have been analyzed for their major oxide and rare earth element (REE) concentrations. The samples consist of (1) the fine fraction of sediment, mostly clay material, (2) phosphatic pellets and fish debris, which are dispersed throughout the fine-grained sediment, (3) tabular-shaped phosphatic crusts, which occur within the uppermost few centimeters of sediment, and (4) phosphatic nodules, which occur on the seafloor. The bulk REE concentrations of the concretions suggest that these elements are partitioned between the enclosed detrital material and the apatite fraction. Analysis of the fine-grained sediment with which the samples are associated suggested that this detrital fraction in the concretions should have shale REE values; the analysis of the fish debris suggested that the apatite fraction might have seawater values. The seawater contribution of REE's is negligible in the nodules and crust, in which the apatite occurs as a fine-grained interstitial cement. That is, the concentration of REE's and the REE patterns are predominantly a function of the amount of enclosed fine-grained sediment. By contrast, the REE pattern of the pelletal apatite suggests a seawater source and the absolute REE concentrations are relatively high. The REE P2O5 ratios of the apatite fraction of these samples thus vary from approximately zero (in the case of the crust and nodules) to as much as approximately 1.2 ?? 10-3 (in the case of the pellets). The range of this ratio suggests that rather subtle variations in the depositional environment might cause a significant variation in the REE content of this authigenic fraction of the sediment. Pelletal glauconite was also recovered from one sediment core. Its REE concentrations closely resemble those of the fish debris. ?? 1988.

  13. De Novo Assembly and Transcriptome Analysis of the Rubber Tree (Hevea brasiliensis) and SNP Markers Development for Rubber Biosynthesis Pathways

    PubMed Central

    Mantello, Camila Campos; Cardoso-Silva, Claudio Benicio; da Silva, Carla Cristina; de Souza, Livia Moura; Scaloppi Junior, Erivaldo José; de Souza Gonçalves, Paulo; Vicentini, Renato; de Souza, Anete Pereira

    2014-01-01

    Hevea brasiliensis (Willd. Ex Adr. Juss.) Muell.-Arg. is the primary source of natural rubber that is native to the Amazon rainforest. The singular properties of natural rubber make it superior to and competitive with synthetic rubber for use in several applications. Here, we performed RNA sequencing (RNA-seq) of H. brasiliensis bark on the Illumina GAIIx platform, which generated 179,326,804 raw reads on the Illumina GAIIx platform. A total of 50,384 contigs that were over 400 bp in size were obtained and subjected to further analyses. A similarity search against the non-redundant (nr) protein database returned 32,018 (63%) positive BLASTx hits. The transcriptome analysis was annotated using the clusters of orthologous groups (COG), gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Pfam databases. A search for putative molecular marker was performed to identify simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs). In total, 17,927 SSRs and 404,114 SNPs were detected. Finally, we selected sequences that were identified as belonging to the mevalonate (MVA) and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways, which are involved in rubber biosynthesis, to validate the SNP markers. A total of 78 SNPs were validated in 36 genotypes of H. brasiliensis. This new dataset represents a powerful information source for rubber tree bark genes and will be an important tool for the development of microsatellites and SNP markers for use in future genetic analyses such as genetic linkage mapping, quantitative trait loci identification, investigations of linkage disequilibrium and marker-assisted selection. PMID:25048025

  14. INPP4B and PTEN Loss Leads to PI-3,4-P2 Accumulation and Inhibition of PI3K in TNBC.

    PubMed

    Reed, Darien E; Shokat, Kevan M

    2017-02-14

    Triple-negative breast cancer [TNBC, lacks expression of estrogen receptor (ER), progesterone receptor (PR) and amplification of HER2/Neu] remains one of the most aggressive subtypes, affects the youngest patients and still lacks an effective targeted therapy(1,2). Both phosphatidylinositol-3-kinase (PI3K)-α and -β contribute to oncogenesis of solid tumors, including the development of breast cancer(3). Inositol polyphosphate-4-phosphatase type II (INPP4B) catalyzes the removal of the 4'-phosphate of phosphatidylinositol-(3,4-bisphosphate (PI-3,4-P2) creating phosphatidylinositol-3-phosphate(4). There is debate concerning whether PI-3,4-P2 contributes to Akt and downstream effector activation with the known canonical signaling second messenger, phosphatidylinositol-(3,4,5)-trisphosphate (PIP3) (5-7). If PI-3,4-P2 is a positive effector, INPP4B would be a negative regulator of PI3K signaling and there is some evidence to support this(4,8). Utilizing phosphatase and tensin homolog deleted on chromosome ten (PTEN)-null triple-negative breast tumor cell lines, it was unexpectedly found that silencing INPP4B decreased basal phospho-Akt (pAkt) and cellular proliferation, and in most cases sensitized cells to PI3K-α and -β isoform-specific inhibitors. Conversely, overexpression of INPP4B desensitized cells to PI3K inhibitors in a phosphatase activity-dependent manner. In summary, the current investigation of INPP4B in PTEN-null TNBC suggests new mechanistic insight and the potential for targeted therapy for this aggressive subset of breast cancer.

  15. The Machinery at Endoplasmic Reticulum-Plasma Membrane Contact Sites Contributes to Spatial Regulation of Multiple Legionella Effector Proteins

    PubMed Central

    Hubber, Andree; Arasaki, Kohei; Nakatsu, Fubito; Hardiman, Camille; Lambright, David; De Camilli, Pietro; Nagai, Hiroki; Roy, Craig R.

    2014-01-01

    The Dot/Icm system of the intracellular pathogen Legionella pneumophila has the capacity to deliver over 270 effector proteins into host cells during infection. Important questions remain as to spatial and temporal mechanisms used to regulate such a large array of virulence determinants after they have been delivered into host cells. Here we investigated several L. pneumophila effector proteins that contain a conserved phosphatidylinositol-4-phosphate (PI4P)-binding domain first described in the effector DrrA (SidM). This PI4P binding domain was essential for the localization of effectors to the early L. pneumophila-containing vacuole (LCV), and DrrA-mediated recruitment of Rab1 to the LCV required PI4P-binding activity. It was found that the host cell machinery that regulates sites of contact between the plasma membrane (PM) and the endoplasmic reticulum (ER) modulates PI4P dynamics on the LCV to control localization of these effectors. Specifically, phosphatidylinositol-4-kinase IIIα (PI4KIIIα) was important for generating a PI4P signature that enabled L. pneumophila effectors to localize to the PM-derived vacuole, and the ER-associated phosphatase Sac1 was involved in metabolizing the PI4P on the vacuole to promote the dissociation of effectors. A defect in L. pneumophila replication in macrophages deficient in PI4KIIIα was observed, highlighting that a PM-derived PI4P signature is critical for biogenesis of a vacuole that supports intracellular multiplication of L. pneumophila. These data indicate that PI4P metabolism by enzymes controlling PM-ER contact sites regulate the association of L. pneumophila effectors to coordinate early stages of vacuole biogenesis. PMID:24992562

  16. Atmospheric nutrient inputs to the northern levantine basin from a long-term observation: sources and comparison with riverine inputs

    NASA Astrophysics Data System (ADS)

    Koçak, M.; Kubilay, N.; Tuğrul, S.; Mihalopoulos, N.

    2010-12-01

    Aerosol and rainwater samples have been collected at a rural site located on the coastline of the Eastern Mediterranean, Erdemli, Turkey between January 1999 and December 2007. Riverine sampling was carried out at five Rivers (Ceyhan, Seyhan, Göksu, Berdan and Lamas) draining into the Northeastern Levantine Basin (NLB) between March 2002 and July 2007. Samples have been analyzed for macronutrients of phosphate, silicate, nitrate and ammonium (PO43-, Sidiss, NO3- and NH4+). Phosphate and silicate in aerosol and rainwater showed higher and larger variations during the transitional period when air flows predominantly originate from North Africa and Middle East/Arabian Peninsula. Deficiency of alkaline material have been found to be the main reason of the acidic rain events whilst high pH values (>7) have been associated with high Sidiss concentrations due to sporadic dust events. In general, lowest nitrate and ammonium concentrations in aerosol and rainwater have been associated with air flow from the Mediterranean Sea. Comparison of atmospheric with riverine fluxes demonstrated that DIN and PO43- fluxes to NLB have been dominated by atmosphere (~90% and ~60% respectively) whereas the input of Si was mainly derived from riverine runoff (~90%). N/P ratios in the atmospheric deposition (233); riverine discharge (28) revealed that NLB receives excessive amounts of DIN and this unbalanced P and N inputs may provoke even more phosphorus deficiency. Observed molar Si/N ratio suggested Si limitation relative to nitrogen might cause a switch from diatom dominated communities to non-siliceous populations particularly at coastal NLB.

  17. Dental Composites with Calcium / Strontium Phosphates and Polylysine

    PubMed Central

    Panpisut, Piyaphong; Liaqat, Saad; Zacharaki, Eleni; Xia, Wendy; Petridis, Haralampos; Young, Anne Margaret

    2016-01-01

    Purpose This study developed light cured dental composites with added monocalcium phosphate monohydrate (MCPM), tristrontium phosphate (TSrP) and antimicrobial polylysine (PLS). The aim was to produce composites that have enhanced water sorption induced expansion, can promote apatite precipitation and release polylysine. Materials and Methods Experimental composite formulations consisted of light activated dimethacrylate monomers combined with 80 wt% powder. The powder phase contained a dental glass with and without PLS (2.5 wt%) and/or reactive phosphate fillers (15 wt% TSrP and 10 wt% MCPM). The commercial composite, Z250, was used as a control. Monomer conversion and calculated polymerization shrinkage were assessed using FTIR. Subsequent mass or volume changes in water versus simulated body fluid (SBF) were quantified using gravimetric studies. These were used, along with Raman and SEM, to assess apatite precipitation on the composite surface. PLS release was determined using UV spectroscopy. Furthermore, biaxial flexural strengths after 24 hours of SBF immersion were obtained. Results Monomer conversion of the composites decreased upon the addition of phosphate fillers (from 76 to 64%) but was always higher than that of Z250 (54%). Phosphate addition increased water sorption induced expansion from 2 to 4% helping to balance the calculated polymerization shrinkage of ~ 3.4%. Phosphate addition promoted apatite precipitation from SBF. Polylysine increased the apatite layer thickness from ~ 10 to 20 μm after 4 weeks. The novel composites showed a burst release of PLS (3.7%) followed by diffusion-controlled release irrespective of phosphate addition. PLS and phosphates decreased strength from 154 MPa on average by 17% and 18%, respectively. All formulations, however, had greater strength than the ISO 4049 requirement of > 80 MPa. Conclusion The addition of MCPM with TSrP promoted hygroscopic expansion, and apatite formation. These properties are expected to help

  18. Inhibition of TGF-β signaling in tumor cells by small molecule Src family kinase inhibitors.

    PubMed

    Bartscht, Tobias; Rosien, Benjamin; Rades, Dirk; Kaufmann, Roland; Biersack, Harald; Lehnerta, Hendrik; Ungefroren, Hendrik

    2017-01-02

    In a series of studies carried out over the last couple of years in various cell types, it was observed that the experimentally used Src family kinase inhibitors PP1 and PP2 and the clinically used Src/Abl inhibitors AZM475271 and dasatinib are potent inhibitors of TGF-β mediated cellular responses such as Smad and p38 mitogen-activated protein kinase phosphorylation, Smad-dependent transcriptional activation, growth inhibition, epithelial-mesenchymal transition (EMT), and cell motility. While for PP1/PP2 it was demonstrated shown that these agents directly inhibit the kinase activity of the TGF-β type I receptor activin receptor-like kinase 5, the mechanism of the anti-TGF-β effect of AZM475271 and dasatinib is less clear. In contrast, the anti-TGF-β effect of yet another Src/Abl inhibitor, bosutinib, is more variable with respect to the type of the TGF-β response and the cell type affected, and lacks a clear dose-dependency. In the light of their strong anti-activin receptor-like kinase 5 kinase effect, PP1 and PP2 should not be used when studying the role of c-Src as downstream mediators in TGF-β/activin receptor-like kinase 5 signaling. On the other hand, based upon in vitro findings, it is conceivable that part of the therapeutic effects of AZM475271 and dasatinib seen in preclinical and clinical studies with solid tumors was caused by inhibition of prometastatic TGF-β rather than Src signaling. If AZM475271 and dasatinib can indeed act as dual Src / TGF-β inhibitors in vivo, this may be beneficial for prevention of metastatic disease in more advanced tumor stages.

  19. Characterization of FAB1 phosphatidylinositol kinases in Arabidopsis pollen tube growth and fertilization.

    PubMed

    Serrazina, Susana; Dias, Fernando Vaz; Malhó, Rui

    2014-08-01

    In yeast and animal cells, phosphatidylinositol-3-monophosphate 5-kinases produce phosphatidylinositol (3,5)-bisphosphate (PtdIns(3,5)P2) and have been implicated in endomembrane trafficking and pH control in the vacuole. In plants, PtdIns(3,5)P2 is synthesized by the Fab1 family, four orthologs of which exist in Arabidopsis: FAB1A and FAB1B, both from the PIKfyve/Fab1 family; FAB1C and FAB1D, both without a PIKfyve domain and of unclear role. Using a reverse genetics and cell biology approach, we investigated the function of the Arabidopsis genes encoding FAB1B and FAB1D, both highly expressed in pollen. Pollen viability, germination and tube morphology were not significantly affected in homozygous mutant plants. In vivo, mutant pollen fertilized ovules leading to normal seeds and siliques. The same result was obtained when mutant ovules were fertilized with wild-type pollen. Double mutant pollen for the two genes was able to fertilize and develop plants no different from the wild-type. At the cellular level, fab1b and fab1d pollen tubes were found to exhibit perturbations in membrane recycling, vacuolar acidification and decreased production of reactive oxygen species (ROS). Subcellular imaging of FAB1B-GFP revealed that the protein localized to the endomembrane compartment, whereas FAB1D-GFP localized mostly to the cytosol and sperm cells. These results were discussed considering possible complementary roles of FAB1B and FAB1D.

  20. Kinase impact assessment in the landscape of fusion genes that retain kinase domains: a pan-cancer study.

    PubMed

    Kim, Pora; Jia, Peilin; Zhao, Zhongming

    2016-12-24

    Assessing the impact of kinase in gene fusion is essential for both identifying driver fusion genes (FGs) and developing molecular targeted therapies. Kinase domain retention is a crucial factor in kinase fusion genes (KFGs), but such a systematic investigation has not been done yet. To this end, we analyzed kinase domain retention (KDR) status in chimeric protein sequences of 914 KFGs covering 312 kinases across 13 major cancer types. Based on 171 kinase domain-retained KFGs including 101 kinases, we studied their recurrence, kinase groups, fusion partners, exon-based expression depth, short DNA motifs around the break points and networks. Our results, such as more KDR than 5'-kinase fusion genes, combinatorial effects between 3'-KDR kinases and their 5'-partners and a signal transduction-specific DNA sequence motif in the break point intronic sequences, supported positive selection on 3'-kinase fusion genes in cancer. We introduced a degree-of-frequency (DoF) score to measure the possible number of KFGs of a kinase. Interestingly, kinases with high DoF scores tended to undergo strong gene expression alteration at the break points. Furthermore, our KDR gene fusion network analysis revealed six of the seven kinases with the highest DoF scores (ALK, BRAF, MET, NTRK1, NTRK3 and RET) were all observed in thyroid carcinoma. Finally, we summarized common features of 'effective' (highly recurrent) kinases in gene fusions such as expression alteration at break point, redundant usage in multiple cancer types and 3'-location tendency. Collectively, our findings are useful for prioritizing driver kinases and FGs and provided insights into KFGs' clinical implications.

  1. Dual Targeting of CDK4 and ARK5 Using a Novel Kinase Inhibitor ON123300 Exerts Potent Anticancer Activity against Multiple Myeloma.

    PubMed

    Perumal, Deepak; Kuo, Pei-Yu; Leshchenko, Violetta V; Jiang, Zewei; Divakar, Sai Krishna Athaluri; Cho, Hearn Jay; Chari, Ajai; Brody, Joshua; Reddy, M V Ramana; Zhang, Weijia; Reddy, E Premkumar; Jagannath, Sundar; Parekh, Samir

    2016-03-01

    Multiple myeloma is a fatal plasma cell neoplasm accounting for over 10,000 deaths in the United States each year. Despite new therapies, multiple myeloma remains incurable, and patients ultimately develop drug resistance and succumb to the disease. The response to selective CDK4/6 inhibitors has been modest in multiple myeloma, potentially because of incomplete targeting of other critical myeloma oncogenic kinases. As a substantial number of multiple myeloma cell lines and primary samples were found to express AMPK-related protein kinase 5(ARK5), a member of the AMPK family associated with tumor growth and invasion, we examined whether dual inhibition of CDK4 and ARK5 kinases using ON123300 results in a better therapeutic outcome. Treatment of multiple myeloma cell lines and primary samples with ON123300 in vitro resulted in rapid induction of cell-cycle arrest followed by apoptosis. ON123300-mediated ARK5 inhibition or ARK5-specific siRNAs resulted in the inhibition of the mTOR/S6K pathway and upregulation of the AMPK kinase cascade. AMPK upregulation resulted in increased SIRT1 levels and destabilization of steady-state MYC protein. Furthermore, ON123300 was very effective in inhibiting tumor growth in mouse xenograft assays. In addition, multiple myeloma cells sensitive to ON123300 were found to have a unique genomic signature that can guide the clinical development of ON123300. Our study provides preclinical evidence that ON123300 is unique in simultaneously inhibiting key oncogenic pathways in multiple myeloma and supports further development of ARK5 inhibition as a therapeutic approach in multiple myeloma.

  2. Phosphoinositides regulate membrane-dependent actin assembly by latex bead phagosomes.

    PubMed

    Defacque, Hélène; Bos, Evelyne; Garvalov, Boyan; Barret, Cécile; Roy, Christian; Mangeat, Paul; Shin, Hye-Won; Rybin, Vladimir; Griffiths, Gareth

    2002-04-01

    Actin assembly on membrane surfaces is an elusive process in which several phosphoinositides (PIPs) have been implicated. We have reconstituted actin assembly using a defined membrane surface, the latex bead phagosome (LBP), and shown that the PI(4,5)P(2)-binding proteins ezrin and/or moesin were essential for this process (). Here, we provide several lines of evidence that both preexisting and newly synthesized PI(4,5)P(2), and probably PI(4)P, are essential for phagosomal actin assembly; only these PIPs were routinely synthesized from ATP during in vitro actin assembly. Treatment of LBP with phospholipase C or with adenosine, an inhibitor of type II PI 4-kinase, as well as preincubation with anti-PI(4)P or anti-PI(4,5)P(2) antibodies all inhibited this process. Incorporation of extra PI(4)P or PI(4,5)P(2) into the LBP membrane led to a fivefold increase in the number of phagosomes that assemble actin. An ezrin mutant mutated in the PI(4,5)P(2)-binding sites was less efficient in binding to LBPs and in reconstituting actin assembly than wild-type ezrin. Our data show that PI 4- and PI 5-kinase, and under some conditions also PI 3-kinase, activities are present on LBPs and can be activated by ATP, even in the absence of GTP or cytosolic components. However, PI 3-kinase activity is not required for actin assembly, because the process was not affected by PI 3-kinase inhibitors. We suggest that the ezrin-dependent actin assembly on the LBP membrane may require active turnover of D4 and D5 PIPs on the organelle membrane.

  3. Molecular definition of a novel inositol polyphosphate metabolic pathway initiated by inositol 1,4,5-trisphosphate 3-kinase activity in Saccharomyces cerevisiae.

    PubMed

    Seeds, Andrew M; Bastidas, Robert J; York, John D

    2005-07-29

    The production of inositol polyphosphate (IPs) and pyrophosphates (PP-IPs) from inositol 1,4,5-trisphosphate (I(1,4,5)P3) requires the 6-/3-/5-kinase activity of Ipk2 (also known as Arg82 and inositol polyphosphate multikinase). Here, we probed the distinct roles for I(1,4,5)P3 6- versus 3-kinase activities in IP metabolism and cellular functions reported for Ipk2. Expression of either I(1,4,5)P3 6- or 3-kinase activity rescued growth of ipk2-deficient yeast at high temperatures, whereas only 6-kinase activity enabled growth on ornithine as the sole nitrogen source. Analysis of IP metabolism revealed that the 3-kinase initiated the synthesis of novel pathway consisting of over eleven IPs and PP-IPs. This pathway was present in wild-type and ipk2 null cells, albeit at low levels as compared with inositol hexakisphosphate synthesis. The primary route of synthesis was: I(1,4,5)P3 --> I(1,3,4,5)P4 --> I(1,2,3,4,5)P5 --> PP-IP4 --> PP2-IP3 and required Kcs1 (or possibly Ipk2), Ipk1, a novel inositol pyrophosphate synthase, and then Kcs1 again, respectively. Mutation of kcs1 ablated this pathway in ipk2 null cells and overexpression of Kcs1 in ipk2 mutant cells phenocopied IP3K expression, confirming it harbors a novel 3-kinase activity. Our work provides a revised genetic map of IP metabolism in yeast and evidence for dosage compensation between IPs and PP-IPs downstream of I(1,4,5)P3 in the regulation of nucleocytoplasmic processes.

  4. Up-regulation of phosphoinositide metabolism in tobacco cells constitutively expressing the human type I inositol polyphosphate 5-phosphatase

    NASA Technical Reports Server (NTRS)

    Perera, Imara Y.; Love, John; Heilmann, Ingo; Thompson, William F.; Boss, Wendy F.; Brown, C. S. (Principal Investigator)

    2002-01-01

    To evaluate the impact of suppressing inositol 1,4,5-trisphosphate (InsP(3)) in plants, tobacco (Nicotiana tabacum) cells were transformed with the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), an enzyme which specifically hydrolyzes InsP(3). The transgenic cell lines showed a 12- to 25-fold increase in InsP 5-ptase activity in vitro and a 60% to 80% reduction in basal InsP(3) compared with wild-type cells. Stimulation with Mas-7, a synthetic analog of the wasp venom peptide mastoparan, resulted in an approximately 2-fold increase in InsP(3) in both wild-type and transgenic cells. However, even with stimulation, InsP(3) levels in the transgenic cells did not reach wild-type basal values, suggesting that InsP(3) signaling is compromised. Analysis of whole-cell lipids indicated that phosphatidylinositol 4,5-bisphosphate (PtdInsP(2)), the lipid precursor of InsP(3), was greatly reduced in the transgenic cells. In vitro assays of enzymes involved in PtdInsP(2) metabolism showed that the activity of the PtdInsP(2)-hydrolyzing enzyme phospholipase C was not significantly altered in the transgenic cells. In contrast, the activity of the plasma membrane PtdInsP 5 kinase was increased by approximately 3-fold in the transgenic cells. In vivo labeling studies revealed a greater incorporation of (32)P into PtdInsP(2) in the transgenic cells compared with the wild type, indicating that the rate of PtdInsP(2) synthesis was increased. These studies show that the constitutive expression of the human type I InsP 5-ptase in tobacco cells leads to an up-regulation of the phosphoinositide pathway and highlight the importance of PtdInsP(2) synthesis as a regulatory step in this system.

  5. Functional interaction of Rpb1 and Spt5 C-terminal domains in co-transcriptional histone modification

    PubMed Central

    Mbogning, Jean; Pagé, Viviane; Burston, Jillian; Schwenger, Emily; Fisher, Robert P.; Schwer, Beate; Shuman, Stewart; Tanny, Jason C.

    2015-01-01

    Transcription by RNA polymerase II (RNAPII) is accompanied by a conserved pattern of histone modifications that plays important roles in regulating gene expression. The establishment of this pattern requires phosphorylation of both Rpb1 (the largest RNAPII subunit) and the elongation factor Spt5 on their respective C-terminal domains (CTDs). Here we interrogated the roles of individual Rpb1 and Spt5 CTD phospho-sites in directing co-transcriptional histone modifications in the fission yeast Schizosaccharomyces pombe. Steady-state levels of methylation at histone H3 lysines 4 (H3K4me) and 36 (H3K36me) were sensitive to multiple mutations of the Rpb1 CTD repeat motif (Y1S2P3T4S5P6S7). Ablation of the Spt5 CTD phospho-site Thr1 reduced H3K4me levels but had minimal effects on H3K36me. Nonetheless, Spt5 CTD mutations potentiated the effects of Rpb1 CTD mutations on H3K36me, suggesting overlapping functions. Phosphorylation of Rpb1 Ser2 by the Cdk12 orthologue Lsk1 positively regulated H3K36me but negatively regulated H3K4me. H3K36me and histone H2B monoubiquitylation required Rpb1 Ser5 but were maintained upon inactivation of Mcs6/Cdk7, the major kinase for Rpb1 Ser5 in vivo, implicating another Ser5 kinase in these regulatory pathways. Our results elaborate the CTD ‘code’ for co-transcriptional histone modifications. PMID:26275777

  6. PIKfyve Regulation of Endosome-Linked Pathways

    PubMed Central

    de Lartigue, Jane; Polson, Hannah; Feldman, Morri; Shokat, Kevan; Tooze, Sharon A; Urbé, Sylvie; Clague, Michael J

    2009-01-01

    The phosphoinositide 5-kinase (PIKfyve) is a critical enzyme for the synthesis of PtdIns(3,5)P2, that has been implicated in various trafficking events associated with the endocytic pathway. We have now directly compared the effects of siRNA-mediated knockdown of PIKfyve in HeLa cells with a specific pharmacological inhibitor of enzyme activity. Both approaches induce changes in the distribution of CI-M6PR and trans-Golgi network (TGN)-46 proteins, which cycles between endosomes and TGN, leading to their accumulation in dispersed punctae, whilst the TGN marker golgin-245 retains a perinuclear disposition. Trafficking of CD8-CI-M6PR (retromer-dependent) and CD8-Furin (retromer-independent) chimeras from the cell surface to the TGN is delayed following drug administration, as is the transport of the Shiga toxin B-subunit. siRNA knockdown of PIKfyve produced no defect in epidermal growth factor receptor (EGFR) degradation, unless combined with knockdown of its activator molecule Vac14, suggesting that a low threshold of PtdIns(3,5)P2 is necessary and sufficient for this pathway. Accordingly pharmacological inhibition of PIKfyve results in a profound block to the lysosomal degradation of activated epidermal growth factor (EGF) and Met receptors. Immunofluorescence revealed EGF receptors to be trapped in the interior of a swollen endosomal compartment. In cells starved of amino acids, PIKfyve inhibition leads to the accumulation of the lipidated form of GFP-LC3, a marker of autophagosomal structures, which can be visualized as fluorescent punctae. We suggest that PIKfyve inhibition may render the late endosome/lysosome compartment refractory to fusion with both autophagosomes and with EGFR-containing multivesicular bodies. PMID:19582903

  7. The Ionic Environment Controls the Contribution of the Barley HvHAK1 Transporter to Potassium Acquisition1[W][OA

    PubMed Central

    Fulgenzi, Fabiana R.; Peralta, María Luisa; Mangano, Silvina; Danna, Cristian H.; Vallejo, Augusto J.; Puigdomenech, Pere; Santa-María, Guillermo E.

    2008-01-01

    The control of potassium (K+) acquisition is a critical requirement for plant growth. Although HAK1 (high affinity K+ 1) transporters provide a pathway for K+ acquisition, the effect exerted by the ionic environment on their contribution to K+ capture remains essentially unknown. Here, the influence of the ionic environment on the accumulation of transcripts coding for the barley (Hordeum vulgare) HvHAK1 transporter as well as on HvHAK1-mediated K+ capture has been examined. In situ mRNA hybridization studies show that HvHAK1 expression occurs in most root cells, being augmented at the outermost cell layers. Accumulation of HvHAK1 transcripts is enhanced by K+ deprivation and transiently by exposure to high salt concentrations. In addition, studies on the accumulation of transcripts coding for HvHAK1 and its close homolog HvHAK1b revealed the presence of two K+-responsive pathways, one repressed and the other insensitive to ammonium. Experiments with Arabidopsis (Arabidopsis thaliana) HvHAK1-expressing transgenic plants showed that K+ deprivation enhances the capture of K+ mediated by HvHAK1. A detailed study with HvHAK1-expressing Saccharomyces cerevisiae cells also revealed an increase of K+ uptake after K+ starvation. This increase did not occur in cells grown at high Na+ concentrations but took place for cells grown in the presence of NH4+. 3,3′-Dihexyloxacarbocyanine iodide accumulation measurements indicate that the increased capture of K+ in HvHAK1-expressing yeast cells cannot be explained only by changes in the membrane potential. It is shown that the yeast protein phosphatase PPZ1 as well as the halotolerance HAL4/HAL5 kinases negatively regulate the HvHAK1-mediated K+ transport. PMID:18359846

  8. Cyclin-dependent kinase 5 regulates degranulation in human eosinophils.

    PubMed

    Odemuyiwa, Solomon O; Ilarraza, Ramses; Davoine, Francis; Logan, Michael R; Shayeganpour, Anooshirvan; Wu, Yingqi; Majaesic, Carina; Adamko, Darryl J; Moqbel, Redwan; Lacy, Paige

    2015-04-01

    Degranulation from eosinophils in response to secretagogue stimulation is a regulated process that involves exocytosis of granule proteins through specific signalling pathways. One potential pathway is dependent on cyclin-dependent kinase 5 (Cdk5) and its effector molecules, p35 and p39, which play a central role in neuronal cell exocytosis by phosphorylating Munc18, a regulator of SNARE binding. Emerging evidence suggests a role for Cdk5 in exocytosis in immune cells, although its role in eosinophils is not known. We sought to examine the expression of Cdk5 and its activators in human eosinophils, and to assess the role of Cdk5 in eosinophil degranulation. We used freshly isolated human eosinophils and analysed the expression of Cdk5, p35, p39 and Munc18c by Western blot, RT-PCR, flow cytometry and immunoprecipitation. Cdk5 kinase activity was determined following eosinophil activation. Cdk5 inhibitors were used (roscovitine, AT7519 and small interfering RNA) to determine its role in eosinophil peroxidase (EPX) secretion. Cdk5 was expressed in association with Munc18c, p35 and p39, and phosphorylated following human eosinophil activation with eotaxin/CCL11, platelet-activating factor, and secretory IgA-Sepharose. Cdk5 inhibitors (roscovitine, AT7519) reduced EPX release when cells were stimulated by PMA or secretory IgA. In assays using small interfering RNA knock-down of Cdk5 expression in human eosinophils, we observed inhibition of EPX release. Our findings suggest that in activated eosinophils, Cdk5 is phosphorylated and binds to Munc18c, resulting in Munc18c release from syntaxin-4, allowing SNARE binding and vesicle fusion, with subsequent eosinophil degranulation. Our work identifies a novel role for Cdk5 in eosinophil mediator release by agonist-induced degranulation.

  9. The Voltage Activation of Cortical KCNQ Channels Depends on Global PIP2 Levels

    PubMed Central

    Kim, Kwang S.; Duignan, Kevin M.; Hawryluk, Joanna M.; Soh, Heun; Tzingounis, Anastasios V.

    2016-01-01

    The slow afterhyperpolarization (sAHP) is a calcium-activated potassium conductance with critical roles in multiple physiological processes. Pharmacological and genetic data suggest that KCNQ channels partly mediate the sAHP. However, these channels are not typically open within the observed voltage range of the sAHP. Recent work has shown that the sAHP is gated by increased PIP2 levels, which are generated downstream of calcium binding by neuronal calcium sensors such as hippocalcin. Here, we examined whether changes in PIP2 levels could shift the voltage-activation range of KCNQ channels. In HEK293T cells, expression of the PIP5 kinase PIPKIγ90, which increases global PIP2 levels, shifted the KCNQ voltage activation to within the operating range of the sAHP. Further, the sensitivity of this effect on KCNQ3 channels appeared to be higher than that on KCNQ2. Therefore, we predict that KCNQ3 plays an essential role in maintaining the sAHP under low PIP2 conditions. In support of this notion, we find that sAHP inhibition by muscarinic receptors that increase phosphoinositide turnover in neurons is enhanced in Kcnq3-knockout mice. Likewise, the presence of KCNQ3 is essential for maintaining the sAHP when hippocalcin is ablated, a condition that likely impairs PIP2 generation. Together, our results establish the relationship between PIP2 and the voltage dependence of cortical KCNQ channels (KCNQ2/3, KCNQ3/5, and KCNQ5), and suggest a possible mechanism for the involvement of KCNQ channels in the sAHP. PMID:26958886

  10. Transforming growth factor-β1 induces type II collagen and aggrecan expression via activation of extracellular signal-regulated kinase 1/2 and Smad2/3 signaling pathways.

    PubMed

    Zhu, Yanhui; Tao, Hairong; Jin, Chen; Liu, Yonzhang; Lu, Xiongwei; Hu, Xiaopeng; Wang, Xiang

    2015-10-01

    Transforming growth factor (TGF)‑β regulates the anabolic metabolism of articular cartilage and prevents cartilage degradation. TGF‑β1 influences cellular proliferation, differentiation and the extracellular matrix through activation of the extracellular signal‑regulated kinase (ERK)1/2 and Smad2/3 signaling pathways. However, it has remained to be fully elucidated precisely how the ERK1/2 and Smad2/3 signaling pathways mediate anabolic processes of articular cartilage. The present study investigated how ERK1/2 and Smad2/3 signaling mediate TGF‑β1‑stimulated type II collagen and aggrecan expression in rat chondrocytes. The results confirmed that TGF‑β1 stimulates type II collagen and aggrecan expression in rat chondrocytes, and furthermore, that the ERK1/2 and Smad2/3 signaling pathways were activated by TGF‑β1. Conversely, the TGF‑β receptor I (ALK5) kinase inhibitor SB525334 significantly impaired TGF‑β1‑induced type II collagen and aggrecan expression, coinciding with a reduction of ERK1/2 and Smad3 phosphorylation. In addition, TGF‑β1‑induced type II collagen and aggrecan expression were significantly suppressed by ERK1/2 inhibitor PD98059. Similarly, TGF‑β1‑stimulated type II collagen and aggrecan expression were decreased in the presence of a Smad3 phosphorylation inhibitor SIS3. Therefore, the present study demonstrated that the ERK1/2 and Smad2/3 signaling pathways regulate type II collagen and aggrecan expression in rat chondrocytes.

  11. The Protein Complex of Neurodegeneration-related Phosphoinositide Phosphatase Sac3 and ArPIKfyve Binds the Lewy Body-associated Synphilin-1, Preventing Its Aggregation*

    PubMed Central

    Ikonomov, Ognian C.; Sbrissa, Diego; Compton, Lauren M.; Kumar, Rita; Tisdale, Ellen J.; Chen, Xuequn; Shisheva, Assia

    2015-01-01

    The 5-phosphoinositide phosphatase Sac3, in which loss-of-function mutations are linked to neurodegenerative disorders, forms a stable cytosolic complex with the scaffolding protein ArPIKfyve. The ArPIKfyve-Sac3 heterodimer interacts with the phosphoinositide 5-kinase PIKfyve in a ubiquitous ternary complex that couples PtdIns(3,5)P2 synthesis with turnover at endosomal membranes, thereby regulating the housekeeping endocytic transport in eukaryotes. Neuron-specific associations of the ArPIKfyve-Sac3 heterodimer, which may shed light on the neuropathological mechanisms triggered by Sac3 dysfunction, are unknown. Here we conducted mass spectrometry analysis for brain-derived interactors of ArPIKfyve-Sac3 and unraveled the α-synuclein-interacting protein Synphilin-1 (Sph1) as a new component of the ArPIKfyve-Sac3 complex. Sph1, a predominantly neuronal protein that facilitates aggregation of α-synuclein, is a major component of Lewy body inclusions in neurodegenerative α-synucleinopathies. Modulations in ArPIKfyve/Sac3 protein levels by RNA silencing or overexpression in several mammalian cell lines, including human neuronal SH-SY5Y or primary mouse cortical neurons, revealed that the ArPIKfyve-Sac3 complex specifically altered the aggregation properties of Sph1-GFP. This effect required an active Sac3 phosphatase and proceeded through mechanisms that involved increased Sph1-GFP partitioning into the cytosol and removal of Sph1-GFP aggregates by basal autophagy but not by the proteasomal system. If uncoupled from ArPIKfyve elevation, overexpressed Sac3 readily aggregated, markedly enhancing the aggregation potential of Sph1-GFP. These data identify a novel role of the ArPIKfyve-Sac3 complex in the mechanisms controlling aggregate formation of Sph1 and suggest that Sac3 protein deficiency or overproduction may facilitate aggregation of aggregation-prone proteins, thereby precipitating the onset of multiple neuronal disorders. PMID:26405034

  12. The TRPM8 channel forms a complex with the 5-HT(1B) receptor and phospholipase D that amplifies its reversal of pain hypersensitivity.

    PubMed

    Vinuela-Fernandez, Ignacio; Sun, Liting; Jerina, Helen; Curtis, John; Allchorne, Andrew; Gooding, Hayley; Rosie, Roberta; Holland, Pamela; Tas, Basak; Mitchell, Rory; Fleetwood-Walker, Sue

    2014-04-01

    Effective relief from chronic hypersensitive pain states remains an unmet need. Here we report the discovery that the TRPM8 ion channel, co-operating with the 5-HT(1B) receptor (5-HT(1B)R) in a subset of sensory afferents, exerts an influence at the spinal cord level to suppress central hypersensitivity in pain processing throughout the central nervous system. Using cell line models, ex vivo rat neural tissue and in vivo pain models, we assessed functional Ca(2+) fluorometric responses, protein:protein interactions, immuno-localisation and reflex pain behaviours, with pharmacological and molecular interventions. We report 5-HT(1B)R expression in many TRPM8-containing afferents and direct interaction of these proteins in a novel multi-protein signalling complex, which includes phospholipase D1 (PLD1). We provide evidence that the 5-HT(1B)R activates PLD1 to subsequently activate PIP 5-kinase and generate PIP2, an allosteric enhancer of TRPM8, achieving a several-fold increase in potency of TRPM8 activation. The enhanced activation responses of synaptoneurosomes prepared from spinal cord and cortical regions of animals with a chronic inflammatory pain state are inhibited by TRPM8 activators that were applied in vivo topically to the skin, an effect potentiated by co-administered 5-HT(1B)R agonists and attenuated by 5-HT(1B)R antagonists, while 5-HT(1B)R agents alone had no detectable effect. Corresponding results are seen when assessing reflex behaviours in inflammatory and neuropathic pain models. Control experiments with alternative receptor/TRP channel combinations reveal no such synergy. Identification of this novel receptor/effector/channel complex and its impact on nociceptive processing give new insights into possible strategies for enhanced analgesia in chronic pain.

  13. The interaction of Munc 18 (p67) with the p10 domain of p35 protects in vivo Cdk5/p35 activity from inhibition by TFP5, a peptide derived from p35

    PubMed Central

    Amin, Niranjana D.; Zheng, Yali; BK, Binukumar; Shukla, Varsha; Skuntz, Susan; Grant, Philip; Steiner, Joseph; Bhaskar, Manju; Pant, Harish C.

    2016-01-01

    In a series of studies, we have identified TFP5, a truncated fragment of p35, the Cdk5 kinase regulatory protein, which inhibits Cdk5/p35 and the hyperactive Cdk5/p25 activities in test tube experiments. In cortical neurons, however, and in vivo in Alzheimer’s disease (AD) model mice, the peptide specifically inhibits the Cdk5/p25 complex and not the endogenous Cdk5/p35. To account for the selective inhibition of Cdk5/p25 activity, we propose that the “p10” N-terminal domain of p35, absent in p25, spares Cdk5/p35 because p10 binds to macromolecules (e.g., tubulin and actin) as a membrane-bound multimeric complex that favors p35 binding to Cdk5 and catalysis. To test this hypothesis, we focused on Munc 18, a key synapse-associated neuronal protein, one of many proteins copurifying with Cdk5/p35 in membrane-bound multimeric complexes. Here we show that, in vitro, the addition of p67 protects Cdk5/p35 and has no effect on Cdk5/p25 activity in the presence of TFP5. In cortical neurons transfected with p67siRNA, we also show that TFP5 inhibits Cdk5/p35 activity, whereas in the presence of p67 the activity is protected. It does so without affecting any other kinases of the Cdk family of cyclin kinases. This difference may be of significant therapeutic value because the accumulation of the deregulated, hyperactive Cdk5/p25 complex in human brains has been implicated in pathology of AD and other neurodegenerative disorders. PMID:27630261

  14. Study of phase relationships in the Sr{sub 3}(PO{sub 4}){sub 2}–CePO{sub 4} system. Phase diagram and thermal characteristics of phases

    SciTech Connect

    Matraszek, Aleksandra

    2013-07-15

    A diagram representing phase relationships in the Sr{sub 3}(PO{sub 4}){sub 2}–CePO{sub 4} phosphate system has been developed on the basis of results obtained by thermal analysis (DTA/DSC/TGA) and X-ray diffraction (XRD) methods. One intermediate compound with the formula Sr{sub 3}Ce(PO{sub 4}){sub 3} occurs in the Sr{sub 3}(PO{sub 4}){sub 2}–CePO{sub 4} system at temperatures exceeding 1045 °C. The compound has a eulytite structure with the following structural parameters: a=b=c=10.1655(8) Å, α=β=γ=90.00°, V=1050.46(6) Å{sup 3}. It's melting point exceeds 1950 °C. A limited solid solution exists in the system, which possesses the structure of a low-temperature form of Sr{sub 3}(PO{sub 4}){sub 2}. At 1000 °C the maximal concentration of CePO{sub 4} in the solid solution is below 20 mol%. The solid solution phase field narrows with increased temperature. There is a eutectic point in the (Sr{sub 3}(PO{sub 4}){sub 2}+Sr{sub 3}Ce(PO{sub 4}){sub 3}) phase field at 1765 °C and 15 mol% of CePO{sub 4}. The melting temperature of Sr{sub 3}(PO{sub 4}){sub 2} is 1882±15 °C. - Graphical abstract: The phase diagram of Sr{sub 3}(PO{sub 4}){sub 2}–CePO{sub 4} system showing the stability ranges of limited solid solution and Sr{sub 3}Ce(PO{sub 4}){sub 3} phases. - Highlights: • Sr{sub 3}(PO{sub 4}){sub 2} melts at 1882 °C. • Phase diagram of Sr{sub 3}(PO{sub 4}){sub 2}–CePO{sub 4} system has been proposed. • Limited solid solution of CePO{sub 4} in Sr{sub 3}(PO{sub 4}){sub 2} forms in the system. • The Sr{sub 3}Ce(PO{sub 4}){sub 2} phosphate is stable at temperatures above 1045 °C.

  15. Gastro-resistant characteristics of GRAS-grade enteric coatings for pharmaceutical and nutraceutical products.

    PubMed

    Czarnocka, Justyna K; Alhnan, Mohamed A

    2015-01-01

    The use of naturally derived excipients to develop enteric coatings offers significant advantages over conventional synthetic polymers. Unlike synthetic polymers, they are biodegradable, relatively abundant, have no daily intake limits or restrictions on use for dietary and nutraceutical products. However, little information is available on their dissolution properties under different gastrointestinal conditions and in comparison to each other. This work investigated the gastric resistance properties of commercially available GRAS-based coating technologies. Three coating systems were evaluated: ethyl cellulose+carboxymethyl cellulose (EC-CMC), ethyl cellulose+sodium alginate (EC-Alg) and shellac+sodium alginate (Sh-Alg) combinations. The minimum coating levels were optimized to meet USP pharmacopoeial criteria for delayed release formulations (<10% release after 2h in pH 1.2 followed by >80% release after 45 min of pH change). Theophylline 150 mg tablets were coated with 6.5%, 7%, and 2.75% coating levels of formulations EC-CMC, EC-Alg and Sh-Alg, respectively. In vitro dissolution test revealed a fast release in pH 6.8 for ethyl cellulose based coatings: t80% value of 65 and 45 min for EC-CMC and EC-Alg respectively, while a prolonged drug release from Sh-Alg coating was observed in both pH 6.8 and 7.4 phosphate buffers. However, when more biologically relevant bicarbonate buffer was used, all coatings showed slower drug release. Disintegration test, carried out in both simulated gastric and intestinal fluid, confirmed good mechanical resistance of EC-CMC and EC-Alg coating, and revealed poor durability of the thinner Sh-Alg. Under elevated gastric pH conditions (pH 2, 3 and 4), EC-CMC and EC-Alg coatings were broken after 70, 30, 55 min and after 30, 15, 15 min, respectively, while Sh-Alg coated tablets demonstrated gastric resistance at all pH values. In conclusion, none of the GRAS-grade coatings fully complied with the different biological demands of delayed

  16. A Systematic View of the MLO Family in Rice Suggests Their Novel Roles in Morphological Development, Diurnal Responses, the Light-Signaling Pathway, and Various Stress Responses

    PubMed Central

    Nguyen, Van N. T.; Vo, Kieu T. X.; Park, Hyon; Jeon, Jong-Seong; Jung, Ki-Hong

    2016-01-01

    The Mildew resistance Locus O (MLO) family is unique to plants, containing genes that were initially identified as a susceptibility factor to powdery mildew pathogens. However, little is known about the roles and functional diversity of this family in rice, a model crop plant. The rice genome has 12 potential MLO family members. To achieve systematic functional assignments, we performed a phylogenomic analysis by integrating meta-expression data obtained from public sources of microarray data and real-time expression data into a phylogenic tree. Subsequently, we identified 12 MLO genes with various tissue-preferred patterns, including leaf, root, pollen, and ubiquitous expression. This suggested their functional diversity for morphological agronomic traits. We also used these integrated transcriptome data within a phylogenetic context to estimate the functional redundancy or specificity among OsMLO family members. Here, OsMLO12 showed preferential expression in mature pollen; OsMLO4, in the root tips; OsMLO10, throughout the roots except at the tips; and OsMLO8, expression preferential to the leaves and trinucleate pollen. Of particular interest to us was the diurnal expression of OsMLO1, OsMLO3, and OsMLO8, which indicated that they are potentially significant in responses to environmental changes. In osdxr mutants that show defects in the light response, OsMLO1, OsMLO3, OsMLO8, and four calmodulin genes were down-regulated. This finding provides insight into the novel functions of MLO proteins associated with the light-responsive methylerythritol 4-phosphate pathway. In addition, abiotic stress meta-expression data and real-time expression analysis implied that four and five MLO genes in rice are associated with responses to heat and cold stress, respectively. Upregulation of OsMLO3 by Magnaporthe oryzae infection further suggested that this gene participates in the response to pathogens. Our analysis has produced fundamental information that will enhance future

  17. Influence of Mg2+ and pH on n.m.r. spectra and radioligand binding of inositol 1,4,5-trisphosphate.

    PubMed Central

    White, A M; Varney, M A; Watson, S P; Rigby, S; Liu, C S; Ward, J G; Reese, C B; Graham, H C; Williams, R J

    1991-01-01

    We and others have shown that the binding of Ins(1,4,5)P3 to its receptor is pH-sensitive and can be inhibited by Mg2+. In the present study we have used 1H- and 31P-n.m.r. spectroscopy to study whether these effects results from increased ionization of Ins(1,4,5)P3 and a direct interaction with Mg2+ respectively. Under near-physiological conditions of ionic strength (100 mM-KCl), three ionizable groups were observed. The pH titration curve of the 1-phosphate was monophasic, with a pKa of 6.3. The titration curves of the 4- and 5-phosphates were biphasic, suggesting that these groups interact; the pKa values for the 4-phosphate determined by 31P-n.m.r. were 5.7 and 7.8, and for the 5-phosphate they were 5.3 and 7.9. 1H- and 31P-n.m.r. measurements suggest that Mg2+ binds weakly to Ins(1,4,5)P3 at physiological pH. Mg2+ non-competitively inhibited binding of Ins(1,4,5)P3 to its receptor in rat cerebellum and bovine adrenal cortex. Inhibition curves for rat cerebellum at pH 7.1 and 8.5, and also for bovine adrenal cortex at pH 8.5, appeared to be monophasic, with IC50 values (concn. of displacer giving 50% inhibition of specific binding) of 214 microM, 572 microM and 9.1 mM respectively. Scatchard analysis revealed that Mg2+ inhibited binding of Ins(1,4,5)P3 to bovine adrenal cortex at pH 8.5 in a non-competitive manner. Our results suggest that the previously reported pH-sensitivity of the binding of Ins(1,4,5)P3 may be caused by ionization of the phosphate groups in positions 4 and 5, and that the ability of Mg2+ to inhibit the binding of Ins(1,4,5)P3 is not mediated by direct chelation but through a site located on, or close to, the Ins(1,4,5)P3 receptor. Inhibition by Mg2+ is pH-sensitive and can vary at least 10-fold between tissues, suggesting possible receptor heterogeneity. Mg2+ may exert an important regulatory control on the release of Ca2+ by Ins(1,4,5)P3. PMID:1654891

  18. Macromolecular coatings on porous silicon: Applications in drug delivery, biosensing, and composites

    NASA Astrophysics Data System (ADS)

    Perelman, Loren Avery

    Two classes of macromolecules, proteins and polymers, are coated onto porous Si films in a variety of geometries in order to study fundamental behaviors of these coatings and their potential device applications. The unique preparation control that porous Si allows in both nano-morphology and surface functionalization provides the means for the coatings. In chapter two, a drug delivery platform using bovine serum albumin (BSA) protein as a stimuli-responsive capping layer on porous Si is described and characterized. It was found that the surface chemistry of the porous Si film has a profound influence on both drug loading capacity and drug release kinetics, providing for control over these drug release variables. The BSA is observed to act as a pH-responsive trigger for the release of vancomycin from the porous Si film. The drug is safely stored in the porous matrix at pH 4 and is released after triggering with pH 7.4 phosphate buffered saline. Chapter three discusses a porous SiO2-based biosensor that is prepared by oxidizing a porous Si film, adsorbing BSA to the surface as a coating, and functionalizing the protein with specific target probes for vancomycin. The BSA was observed to adsorb strongly to the surface, resisting desoprtion in both phosphate buffered saline and triton-X buffer solutions. Quantitative binding information for the tripeptide Ac-L-Lysine-D-Alanine-D-Alanine and vancomycin is determined using the optical properties of the porous Si as a transduction methodology. Chapters four and five describe the fabrication of thermoresponsive and multifunctional nanohybrids, respectively, using stimuli-responsive hydrogels to infiltrate and coat oxidized porous Si films. The optical properties of the porous Si films are used to study the response of the hydrogel phase of the hybrids to a variety of stimuli. The optical changes correspond to previously-described physical changes in the hydrogel phase, and it was determined that this platform provides a

  19. Thermosensitive and Mucoadhesive Sol-Gel Composites of Paclitaxel/Dimethyl-β-Cyclodextrin for Buccal Delivery

    PubMed Central

    Kang, Bong-Seok; Ng, Choon Lian; Davaa, Enkhzaya; Park, Jeong-Sook

    2014-01-01

    The purpose of this study was to develop a buccal paclitaxel delivery system using the thermosensitive polymer Pluronic F127 (PF127) and the mucoadhesive polymer polyethylene oxide (PEO). The anticancer agent paclitaxel is usually used to treat ovarian, breast, and non-small-cell lung cancer. To improve its aqueous solubility, paclitaxel was incorporated into an inclusion complex with (2,6-di-O-methyl)-β-cyclodextrin (DMβCD). The formation of the paclitaxel inclusion complex was evaluated using various techniques, including x-ray diffractometry (XRD), Fourier-transform infrared (FT-IR) spectrophotometry, differential scanning calorimetry (DSC), and scanning electron microscopy (SEM). Hydrogels were prepared using a cold method. Concentrations of 18, 20, and 23% (w/v) PF127 were dissolved in distilled water including paclitaxel and stored overnight in a refrigerator at 4°C. PEO was added at concentrations of 0.1, 0.2, 0.4, 0.8, and 1% (w/v). Each formulation included paclitaxel (0.5 mg/mL). The sol-gel transition temperature of the hydrogels was measured using the tube-inverting method. Drug release from the hydrogels was measured using a Franz diffusion cell containing pH 7.4 phosphate-buffered solution (PBS) buffer at 37°C. The cytotoxicity of each formulation was measured using the MTT assay with a human oral cancer cell (KB cell). The sol-gel transition temperature of the hydrogel decreased when PF127 was present and varied according to the presence of mucoadhesive polymers. The in vitro release was sustained and the release rate was slowed by the addition of the mucoadhesive polymer. The cytotoxicity of the blank formulation was low, although the drug-loaded hydrogel showed acceptable cytotoxicity. The results of our study suggest that the combination of a PF 127-based mucoadhesive hydrogel formulation and inclusion complexes improves the in vitro release and cytotoxic effect of paclitaxel. PMID:25275485

  20. Relative density of urine: methods and clinical significance.

    PubMed

    Pradella, M; Dorizzi, R M; Rigolin, F

    1988-01-01

    The physical properties and chemical composition of urine are highly variable and are determined in large measure by the quantity and the type of food consumed. The specific gravity is the ratio of the density to that of water, and it is dependent on the number and weight of solute particles and on the temperature of the sample. The weight of solute particles is constituted mainly of urea (73%), chloride (5.4%), sodium (5.1%), potassium (2.4%), phosphate (2.0%), uric acid (1.7%), and sulfate (1.3%). Nevertheless, urine osmolality depends only on the number of solute particles. The renal production of maximally concentrated urine and formation of dilute urine may be reduced to two basic elements: (1) generation and maintenance of a renal medullary solute concentration hypertonic to plasma and (2) a mechanism for osmotic equilibration between the inner medulla and the collecting duct fluid. The interaction of the renal medullary countercurrent system, circulating levels of antidiuretic hormone, and thirst regulates water metabolism. Renin, aldosterone, prostaglandins, and kinins also play a role. Clinical estimation of the concentrating and diluting capacity can be performed by relatively simple provocative tests. However, urinary specific gravity after taking no fluids for 12 h overnight should be 1.025 or more, so that the second urine in the morning is a useful sample for screening purposes. Many preservation procedures affect specific gravity measurements. The concentration of solids (or water) in urine can be measured by weighing, hydrometer, refractometry, surface tension, osmolality, a reagent strip, or oscillations of a capillary tube. These measurements are interrelated, not identical. Urinary density measurement is useful to assess the disorders of water balance and to discriminate between prerenal azotemia and acute tubular necrosis. The water balance regulates the serum sodium concentration, therefore disorders are revealed by hypo- and hypernatremia. The

  1. Determination of plasma total homocysteine and cysteine using HPLC with fluorescence detection and an ammonium 7-fluoro-2, 1, 3-benzoxadiazole-4-sulphonate (SBD-F) derivatization protocol optimized for antioxidant concentration, derivatization reagent concentration, temperature and matrix pH.

    PubMed

    Daskalakis, I; Lucock, M D; Anderson, A; Wild, J; Schorah, C J; Levene, M I

    1996-01-01

    A sensitive HPLC-fluorescence method for determining total endogenous plasma homocysteine (Hcy), cysteine (Cys) and cysteinylglycine (Cys-Gly) following derivatization with ammonium 7-fluoro 2,1,3-benzoxadiazole-4-sulphonate (SBD-F) is described. Quantitation utilizes an internal standard, 2-mercaptoethylamine. The derivatization procedure has been optimized for concentration of SBD-F, reducing agent (tributylphosphine) and temperature. Findings indicate that values for plasma determinations vary according to the nature of the matrix in which calibration standards are made up. If quantitation is based on a peak height ratio, then standards should be made up in either pH 7.4 phosphate buffered saline or plasma taking into account the endogenous thiol concentration. These findings are based on calibration data, and 30 plasma samples quantified using thiol standards made up in plasma, pH 7.4 and pH 9.5 buffers. By defining how this matrix/pH effect influences thiol quantitation, it should be possible to make a more meaningful comparison of Hcy measurements between laboratories. The chromatographic separation was investigated at several mobile-phase pH values with the following conditions ascertained to be optimal: a mobile phase consisting of 5% (v/v) acetonitrile in 0.1 M KH2PO4, pH 2.15 was run at a flow rate of 0.5 mL/min. It was used in conjunction with a Supelco LC-18 base deactivated analytical column (150 x 4.6 cm i.d. 3 microM bonded silica). The internal standard and thiols were measured by fluorescence detection at 385 nm excitation and 515 nm emmission. Plasma levels are easily measured in a 100 microL volume. Storage for 2 months at -20 degrees C resulted in no deterioration of thiols. Furthermore, no difference in thiol levels was observed between bloods collected in lithium heparin and EDTA. Collected blood should, however, be separated as soon as possible to avoid red cell metabolism of Hcy which was observed in a case of hyperhomocysteinemia. Once

  2. Carbon and nutrient cycling in the upper water column across the Polar Frontal Zone and Antarctic Circumpolar Current along 170°W

    NASA Astrophysics Data System (ADS)

    Rubin, Stephany I.

    2003-09-01

    Seasonal changes of upper water column chemical properties integrate the effects of the physical, chemical, and biological processes that control the export of carbon to the deep ocean. Between October 1997 and March 1998, several hydrographic cruises were undertaken in the southwestern Pacific sector of the Southern Ocean, as part of the U.S. JGOFS program. On these cruises, the partial pressure of carbon dioxide (pCO2) and concentration of total carbon dioxide (TCO2) dissolved in seawater were determined in surface waters, along with the nutrient concentrations [nitrate (NO3-), nitrite (NO2-), ammonium (NH4+), phosphate (PO4≡), and silicate (Si(OH)4)]. This interval saw the commencement, culmination, and dénouement of phytoplankton blooms, both north and south of the Polar Front (PF). Nutrient utilization, regeneration, and export ratios, and primary production and export, were estimated from seasonal changes in these properties observed in surface waters across the Polar Front. While the biological drawdowns of carbon dioxide and nutrient concentrations in the euphotic zone were greater south of the front, the estimated primary productivity (1.6-2.7 mol C/m2/yr) and export (1.2-1.5 mol C/m2/yr) are comparable. The observed C/N/P ratios vary temporally, as a function of the dominant process, and spatially, as a function of the phytoplankton assemblage. South of the PF, the maximum biological utilization (or new production) C/N/P ratios are 69 ± 2/10.4 ± 0.5/1 while post-regeneration biological utilization (or export) C/N/P ratios are 87 ± 3/12.7 ± 0.5/1. North of the PF the C/N/P ratios are similar to the classic Redfield ratios, 100 ± 5/(14-16.5) ± 0.5/1. Silica/(C, N, and P) ratios are also highly variable temporally, reflecting differences between silica and organic nutrient cycling, and the bio-availability iron. Departures from the "Redfield ratio" arise from excess phosphate uptake by diatoms and the preferential regeneration of phosphate. The

  3. Fish Oil Supplementation in Humans: Effects on Platelet Responses, Phospholipid Composition and Metabolism.

    NASA Astrophysics Data System (ADS)

    Skeaff, Clark Murray

    associated with ( ^3H) PIP_2/( ^3H) PIP was 0.41 in fish oil consumers and 1.14 in olive oil consumers. These results are consistent with a dampened collagen-induced phosphatidylinositol 4 -phosphate kinase activity in platelets of healthy individuals consuming dietary fish oil. This effect may be eicosanoid -related based on work with BW 755C, a dual inhibitor of the cyclooxygenase and lipoxygenase enzymes. The relevance of these findings to the altered production of inositol 1,4,5 trisphosphate remains to be determined.

  4. Oxidation of Framboidal Pyrite as a Mobilization Mechanism During Aquifer Storage and Recovery in the Upper Floridan Aquifer, Southwest Florida

    NASA Astrophysics Data System (ADS)

    Price, R. E.; Pichler, T.

    2002-12-01

    During a recent study conducted at the University of South Florida, core samples were collected from 20 wells located in the southern half of the Southwest Florida Water Management District (SWFWMD), and analyzed to gain a better understanding of arsenic distribution and mineralogy throughout the Oligocene-aged Suwannee Limestone, Upper Floridan Aquifer. The Suwannee is the primary storage zone utilized to date for Aquifer Storage and Recovery (ASR), the process of capturing excess water during the rainy season and storing that water underground until needed. The SWFWMD recently determined that ASR is a critical component for its 20-year strategy to enhance water supply in southwest Florida where, as of January 2002, 26 facilities are in operation and 19 are permitted for construction. The Comprehensive Everglades Restoration Project (CERP) calls for over 330 ASR wells to store 1.7 billion gallons of water per day. This research was conducted because of the discovery of elevated arsenic (As) levels during recovery cycle testing of several ASR facilities throughout southwest Florida. Before any solution or mobilization mechanism can be suggested, the exact mineralogical association of the arsenic must be determined. Possible sources for the arsenic include: 1) sulfide minerals, 2) iron oxyhydroxide coatings, 3) organic material, and/or 4) phosphate nodules. Sampling was carried out with the purpose to sample each core at constant intervals and to collect samples at locations that may have a higher potential for the presence of arsenic, such as fractures, organic material, sulfide-rich zones and iron oxyhydroxide-rich zones. Detailed lithologic descriptions for each sample showed that framboidal pyrite was present, and in fact abundant, throughout the unit, while the iron oxides, organics, and phosphate nodules were uncommon. Total arsenic concentrations for each sample were determined by hydride generation-atomic fluorescence spectrometry (HG-AFS), which showed much

  5. Factors influencing inactivation of Klebsiella pneumoniae by chlorine and chloramine.

    PubMed

    Goel, Sudha; Bouwer, Edward J

    2004-01-01

    Inactivation of Klebsiella pneumoniae cultures by chlorine and chloramine was evaluated under different growth conditions by varying nutrient media dilution, concentrations of essential inorganic nutrients (FeCl3, MgSO4, phosphate, and ammonium salts), and temperature. All inactivation assays were performed at room temperature (22-23 degrees C) and near neutral pH (7.2-7.5). C*T(99.9) values for chlorine increased >20-fold and for chloramine increased 2.6-fold when cells were grown in 100-fold diluted nutrient broth (2NB) solutions (final TOC of 35-40 mg/L). Background levels of Mg: 6.75 x 10(-2) mM and Fe: 3.58 x 10(-5) mM or high levels of FeCl3 (0.01 mM) and MgSO4 (1 mM) during growth resulted in the highest resistances to chlorine with C*T(99.9) values of 13.06 (+/-0.91) and 13.78 (+/-1.97) mg-min/L, respectively. Addition of low levels of FeCl3 (0.001 mM) and MgSO4 (0.1 mM) to K. pneumoniae cultures during growth resulted in the lowest bacterial resistances to inactivation; C*T(99.9) values ranged from 0.28 (+/-0.06) to 1.88 (+/-0.53)mg-min/L in these cultures. Increase in growth temperature from 22.5 degrees C to 35 degrees C for unamended 2NB cultures resulted in a 42-fold decrease in C*T(99.9) values for chlorine. A similar change in temperature resulted in no significant change in C*T(99.9) values for chloramine. These results indicate that inactivation of K. pneumoniae cultures by chlorine was highly sensitive to changes in growth conditions unlike inactivation by chloramine.

  6. Transcriptome analysis of bitter acid biosynthesis and precursor pathways in hop (Humulus lupulus)

    PubMed Central

    2013-01-01

    Background Bitter acids (e.g. humulone) are prenylated polyketides synthesized in lupulin glands of the hop plant (Humulus lupulus) which are important contributors to the bitter flavour and stability of beer. Bitter acids are formed from acyl-CoA precursors derived from branched-chain amino acid (BCAA) degradation and C5 prenyl diphosphates from the methyl-D-erythritol 4-phosphate (MEP) pathway. We used RNA sequencing (RNA-seq) to obtain the transcriptomes of isolated lupulin glands, cones with glands removed and leaves from high α-acid hop cultivars, and analyzed these datasets for genes involved in bitter acid biosynthesis including the supply of major precursors. We also measured the levels of BCAAs, acyl-CoA intermediates, and bitter acids in glands, cones and leaves. Results Transcripts encoding all the enzymes of BCAA metabolism were significantly more abundant in lupulin glands, indicating that BCAA biosynthesis and subsequent degradation occurs in these specialized cells. Branched-chain acyl-CoAs and bitter acids were present at higher levels in glands compared with leaves and cones. RNA-seq analysis showed the gland-specific expression of the MEP pathway, enzymes of sucrose degradation and several transcription factors that may regulate bitter acid biosynthesis in glands. Two branched-chain aminotransferase (BCAT) enzymes, HlBCAT1 and HlBCAT2, were abundant, with gene expression quantification by RNA-seq and qRT-PCR indicating that HlBCAT1 was specific to glands while HlBCAT2 was present in glands, cones and leaves. Recombinant HlBCAT1 and HlBCAT2 catalyzed forward (biosynthetic) and reverse (catabolic) reactions with similar kinetic parameters. HlBCAT1 is targeted to mitochondria where it likely plays a role in BCAA catabolism. HlBCAT2 is a plastidial enzyme likely involved in BCAA biosynthesis. Phylogenetic analysis of the hop BCATs and those from other plants showed that they group into distinct biosynthetic (plastidial) and catabolic (mitochondrial

  7. Effector Vγ9Vδ2 T cells dominate the human fetal γδ T-cell repertoire

    PubMed Central

    Dimova, Tanya; Brouwer, Margreet; Gosselin, Françoise; Tassignon, Joël; Leo, Oberdan; Donner, Catherine; Marchant, Arnaud; Vermijlen, David

    2015-01-01

    γδ T cells are unconventional T cells recognizing antigens via their γδ T-cell receptor (TCR) in a way that is fundamentally different from conventional αβ T cells. γδ T cells usually are divided into subsets according the type of Vγ and/or Vδ chain they express in their TCR. T cells expressing the TCR containing the γ-chain variable region 9 and the δ-chain variable region 2 (Vγ9Vδ2 T cells) are the predominant γδ T-cell subset in human adult peripheral blood. The current thought is that this predominance is the result of the postnatal expansion of cells expressing particular complementary-determining region 3 (CDR3) in response to encounters with microbes, especially those generating phosphoantigens derived from the 2-C-methyl-d-erythritol 4-phosphate pathway of isoprenoid synthesis. However, here we show that, rather than requiring postnatal microbial exposure, Vγ9Vδ2 T cells are the predominant blood subset in the second-trimester fetus, whereas Vδ1+ and Vδ3+ γδ T cells are present only at low frequencies at this gestational time. Fetal blood Vγ9Vδ2 T cells are phosphoantigen responsive and display very limited diversity in the CDR3 of the Vγ9 chain gene, where a germline-encoded sequence accounts for >50% of all sequences, in association with a prototypic CDR3δ2. Furthermore, these fetal blood Vγ9Vδ2 T cells are functionally preprogrammed (e.g., IFN-γ and granzymes-A/K), with properties of rapidly activatable innatelike T cells. Thus, enrichment for phosphoantigen-responsive effector T cells has occurred within the fetus before postnatal microbial exposure. These various characteristics have been linked in the mouse to the action of selecting elements and would establish a much stronger parallel between human and murine γδ T cells than is usually articulated. PMID:25617367

  8. Effect of artificial rearing of piglets on the volume densities of M cells in the tonsils of the soft palate and ileal Peyer's patches.

    PubMed

    Prims, S; Pintens, N; Vergauwen, H; Van Cruchten, S; Van Ginneken, C; Casteleyn, C

    2017-02-01

    The high prolificacy of modern hybrid sows has increased the mean litter size during the last decades. However, rearing large litters is challenging and has increased the use of alternative management strategies such as euthanasia of weak piglets, cross-fostering, supplementing piglets with milk, split-nursing and split-weaning. The latter includes artificial rearing on brooders where piglets have ad libitum access to milk replacer. The effect of this artificial rearing on the immune system of the piglet is the subject of various studies. The present study focused on the M cells in the tonsil of the soft palate and in the ileal Peyer's patch (iPP). These epithelial cells are specialized in antigen sampling and play a pivotal role in the induction of adaptive immune responses. The volume densities of the M cells were assessed by stereological analysis of tissue samples from piglets of 0, 3, 8 and 19days of age. During the first three days, piglets suckled the sow, permitting them to ingest colostrum. At the third day, the piglets were either allowed to continue to suckle the sow or were transferred to brooders. The six experimental groups, each containing six piglets, thus consisted of newborn piglets, 3-day-old sow-suckled piglets, and conventionally and artificially reared piglets of 8 and 19days of age. To identify M cells, tissue samples were immersed in 4% phosphate-buffered paraformaldehyde and paraffin sections were immunohistochemically stained against cytokeratin 18. The volume densities of M cells in both the crypt epithelium of the tonsils of the soft palate and the follicle-associated epithelium of the iPPs did not show any difference between the various age groups of conventionally reared piglets. However, values were twice as high in the iPPs compared to the tonsils of the soft palate. In contrast, a decrease in volume densities of M cells was observed in the iPPs of piglets after they had been transferred to commercial brooders (P=0.05), resulting in

  9. Albumin binding, relaxivity, and water exchange kinetics of the diastereoisomers of MS-325, a gadolinium(III)-based magnetic resonance angiography contrast agent.

    PubMed

    Caravan, Peter; Parigi, Giacomo; Chasse, Jaclyn M; Cloutier, Normand J; Ellison, Jeffrey J; Lauffer, Randall B; Luchinat, Claudio; McDermid, Sarah A; Spiller, Marga; McMurry, Thomas J

    2007-08-06

    The amphiphilic gadolinium complex MS-325 ((trisodium-{(2-(R)-[(4,4-diphenylcyclohexyl) phosphonooxymethyl] diethylenetriaminepentaacetato) (aquo)gadolinium(III)}) is a contrast agent for magnetic resonance angiography (MRA). MS-325 consists of two slowly interconverting diastereoisomers, A and B (65:35 ratio), which can be isolated at pH > 8.5 (TyeklAr, Z.; Dunham, S. U.; Midelfort, K.; Scott, D. M.; Sajiki, H.; Ong, K.; Lauffer, R. B.; Caravan, P.; McMurry, T. J. Inorg. Chem. 2007, 46, 6621-6631). MS-325 binds to human serum albumin (HSA) in plasma resulting in an extended plasma half-life, retention of the agent within the blood compartment, and an increased relaxation rate of water protons in plasma. Under physiological conditions (37 degrees C, pH 7.4, phosphate buffered saline (PBS), 4.5% HSA, 0.05 mM complex), there is no statistical difference in HSA affinity or relaxivity between the two isomers (A 88.6 +/- 0.6% bound, r1 = 42.0 +/- 1.0 mM(-1) s(-1) at 20 MHz; B 90.2 +/- 0.6% bound, r1 = 38.3 +/- 1.0 mM(-1) s(-1) at 20 MHz; errors represent 1 standard deviation). At lower temperatures, isomer A has a higher relaxivity than isomer B. The water exchange rates in the absence of HSA at 298 K, kA298 = 5.9 +/- 2.8 x 10(6) s(-1), kB298 = 3.2 +/- 1.8 x 10(6) s(-1), and heats of activation, DeltaHA = 56 +/- 8 kJ/mol, DeltaHB = 59 +/- 11 kJ/mol, were determined by variable-temperature 17O NMR at 7.05 T. Proton nuclear magnetic relaxation dispersion (NMRD) profiles were recorded over the frequency range of 0.01-50 MHz at 5, 15, 25, and 35 degrees C in a 4.5% HSA in PBS solution for each isomer (0.1 mM). Differences in the relaxivity in HSA between the two isomers could be attributed to the differing water exchange rates.

  10. Comparative glandular trichome transcriptome based gene characterization reveals reasons for differential (-)-menthol biosynthesis in Mentha species.

    PubMed

    Akhtar, Md Qussen; Qamar, Nida; Yadav, Pallavi; Kulkarni, Pallavi; Kumar, Ajay; Shasany, Ajit Kumar

    2017-02-11

    , isopulegone isomerase; IPR, isopiperitenone reductase; L3H, limonene 3-hydroxylase; LS, limonene synthase; MCS, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; MCT, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase; MD, menthol dehydrogenase; MEP, methylerythritol phosphate; MFS, menthofuran synthase; MVA, mevalonic acid; MVK, mevalonate kinase; NMD, neomenthol dehydrogenase, Nr, non-redundant; PMD, phosphomevalonate decarboxylase; PMK, phosphomevalonate kinase; PR, pulegone reductase; qRT-PCR, quantitative real-time polymerase chain reaction; SSR, simple sequence repeat; TPS, terpene synthase.

  11. Chemical composition of halophytes from the Neusiedler Lake region in Austria.

    PubMed

    Albert, R; Popp, Marianne

    1977-06-01

    The ionic relations in halophytes from the region east of Neusiedler Lake in Austria have been investigated. The study encompasses the following compounds: Na, K, Mg, Ca; Cl, SO4, phosphate, nitrate, and organic acids.The ionic composition varies substantially among the species investigated. Frequently a specific pattern of ion content can be found within a specific taxon. a) Dicotyledons: Extraordinary accumulation of sodium, high intake of inorganic ions (mainly Cl, less SO4), and regular occurrence of free oxalate, causing low Ca-concentrations, are typical for Chenopodiaceae and Caryophyllaceae (Spergularia media). Lepidium crassifolium shows similar sodium preponderance accompanied by high levels of SO4, Cl, and organic anions other than oxalate (mainly citrate and malate). The remaining dicotyledons show rather moderate salt content; Asteraceae and Cichoriaceae prefer Cl, and Plantago maritima accumulates high amounts of SO4 as well as Cl. Malate and citrate are, without exception, the main organic anions. The K:Na ratios in dicotyledons (esp. Chenopodiaceae and Lepidium-Brassicaceae) lie far below unity. b) Monocotyledons: In marked contrast, Poaceae, Cyperaceae, and Juncaceae are characterized by a general low salt status. With few exceptions, Cl is stored as the main inorganic anion, phosphate reaches higher levels than in dicotyledons and in many cases lies in nearly the same concentration range as SO4. The pattern of organic anions with malate and citrate as the main acids, does not basically differ from nonhalophilous species. In any case, K:Na ratio exceeds unity. Triglochin maritimum is the only monocotyle species exhibiting as high salt content and low K:Na ratios as dicotyledons. Nitrate and phosphate are of minor quantitative importance with regard to their osmotic efficiency; their mEq percentage of the total anion concentration range between 0.03 to 2.6 (NO3) and 0.5 to 13.6 (phosphate), respectively.The results are discussed from different

  12. A special acyl carrier protein for transferring long hydroxylated fatty acids to lipid A in Rhizobium.

    PubMed

    Brozek, K A; Carlson, R W; Raetz, C R

    1996-12-13

    Lipid A, the hydrophobic anchor of lipopolysaccharides in the outer membranes of Gram-negative bacteria, varies in structure among different Rhizobiaceae. The Rhizobium meliloti lipid A backbone, like that of Escherichia coli, is a beta1'-6-linked glucosamine disaccharide that is phosphorylated at positions 1 and 4'. Rhizobium leguminosarum lipid A lacks both phosphates, but contains aminogluconate in place of the proximal glucosamine 1-phosphate, and galacturonic acid instead of the 4'-phosphate. A peculiar feature of the lipid As of all Rhizobiaceae is acylation with 27-hydroxyoctacosanoic acid, a long hydroxylated fatty acid not found in E. coli. We now describe an in vitro system, consisting of a membrane enzyme and a cytosolic acyl donor from R. leguminosarum, that transfers 27-hydroxyoctacosanoic acid to (Kdo)2-lipid IVA, a key lipid A precursor common to both E. coli and R. leguminosarum. The 27-hydroxyoctacosanoic acid moiety was detected in the lipid product by mass spectrometry. The membrane enzyme required the presence of Kdo residues in the acceptor substrate for activity. The cytosolic acyl donor was purified from wild-type R. leguminosarum using the acylation of (Kdo)2-[4'-32P]-lipid IVA as the assay. Amino-terminal sequencing of the purified acyl donor revealed an exact 19-amino acid match with a partially sequenced gene (orf*) of R. leguminosarum. Orf* contains the consensus sequence, DSLD, for attachment of 4'-phosphopantetheine. When the entire orf* gene was sequenced, it was found to encode a protein of 92 amino acids. Orf* is a new kind of acyl carrier protein because it is only approximately 25% identical both to the constitutive acyl carrier protein (AcpP) and to the inducible acyl carrier protein (NodF) of R. leguminosarum. Mass spectrometry of purified active Orf* confirmed the presence of 4'-phosphopantetheine and 27-hydroxyoctacosanoic acid in the major species. Smaller mass peaks indicative of Orf* acylation with hydroxylated 20, 22, 24

  13. Evidence of addiction by anesthesiologists as documented by hair analysis.

    PubMed

    Kintz, P; Villain, M; Dumestre, V; Cirimele, V

    2005-10-04

    /liquid extraction after incubation in pH 8.4 phosphate buffer. Fentanyl derivatives are analyzed by GC-MS/MS. The following cases are included in this paper: Case 1: 50-year-old anesthetist, positive for fentanyl (644 pg/mg); Case 2: 42-year-old anesthetist, positive for fentanyl (101 pg/mg) and sufentanil (2 pg/mg); Case 3: 40-year-old anesthetist, positive for codeine (210 pg/mg), alfentanil (30 pg/mg) and midazolam (160 pg/mg); Case 4: 46-year-old nurse, found dead, positive for alfentanil (2 pg/mg) and fentanyl (8 pg/mg). In these cases, the combination of an alternative specimen (hair) and hyphenated analytical techniques (tandem mass spectrometry) appears to be a pre-requisite.

  14. Plastoquinone and Ubiquinone in Plants: Biosynthesis, Physiological Function and Metabolic Engineering

    PubMed Central

    Liu, Miaomiao; Lu, Shanfa

    2016-01-01

    Plastoquinone (PQ) and ubiquinone (UQ) are two important prenylquinones, functioning as electron transporters in the electron transport chain of oxygenic photosynthesis and the aerobic respiratory chain, respectively, and play indispensable roles in plant growth and development through participating in the biosynthesis and metabolism of important chemical compounds, acting as antioxidants, being involved in plant response to stress, and regulating gene expression and cell signal transduction. UQ, particularly UQ10, has also been widely used in people’s life. It is effective in treating cardiovascular diseases, chronic gingivitis and periodontitis, and shows favorable impact on cancer treatment and human reproductive health. PQ and UQ are made up of an active benzoquinone ring attached to a polyisoprenoid side chain. Biosynthesis of PQ and UQ is very complicated with more than thirty five enzymes involved. Their synthetic pathways can be generally divided into two stages. The first stage leads to the biosynthesis of precursors of benzene quinone ring and prenyl side chain. The benzene quinone ring for UQ is synthesized from tyrosine or phenylalanine, whereas the ring for PQ is derived from tyrosine. The prenyl side chains of PQ and UQ are derived from glyceraldehyde 3-phosphate and pyruvate through the 2-C-methyl-D-erythritol 4-phosphate pathway and/or acetyl-CoA and acetoacetyl-CoA through the mevalonate pathway. The second stage includes the condensation of ring and side chain and subsequent modification. Homogentisate solanesyltransferase, 4-hydroxybenzoate polyprenyl diphosphate transferase and a series of benzene quinone ring modification enzymes are involved in this stage. PQ exists in plants, while UQ widely presents in plants, animals and microbes. Many enzymes and their encoding genes involved in PQ and UQ biosynthesis have been intensively studied recently. Metabolic engineering of UQ10 in plants, such as rice and tobacco, has also been tested. In this

  15. Survival of microencapsulated probiotic Lactobacillus paracasei LBC-1e during manufacture of Mozzarella cheese and simulated gastric digestion.

    PubMed

    Ortakci, F; Broadbent, J R; McManus, W R; McMahon, D J

    2012-11-01

    An erythromycin-resistant strain of probiotic Lactobacillus paracasei ssp. paracasei LBC-1 (LBC-1e) was added to part-skim Mozzarella cheese in alginate-microencapsulated or free form at a level of 10(8) and 10(7)cfu/g, respectively. Survival of LBC-1e and total lactic acid bacteria (LAB) was investigated through the pasta filata process of cheese making (in which the cheese curd was heated to 55 °C and stretched in 70 °C-hot brine), followed by storage at 4 °C for 6 wk and simulated gastric and intestinal digestion. This included incubation in 0.1 M and 0.01 M HCl, 0.9 M H(3)PO(4), and a simulated intestinal juice consisting of pancreatin and bile salts in a pH 7.4 phosphate buffer. Some reductions were observed in both free and encapsulated LBC-1e during heating and stretching, with encapsulated LBC-1e surviving slightly better. Changes in total LAB losses during heating and stretching did not reach statistical significance. During storage, a decrease was observed in total LAB, but no statistically significant decrease was observed in LBC-1e. Survival during gastric digestion in HCl was dependent on the extent of neutralization of HCl by the cheese, with more survival in the weaker acid, in which pH increased to 4.4 after cheese addition. The alginate microcapsules did not provide any protection against the HCl. It is interesting that survival of the encapsulated LBC-1e was greater during incubation in H(3)PO(4) than in the HCl gastric juices. Proper selection of simulated gastric digestion media is important for predicting the delivery of probiotic bacteria into the human intestinal tract. Neither free nor encapsulated LBC-1e was affected by incubation in the pancreatin-bile solution. Based on the level of probiotic bacteria in cheese needed to provide a health benefit and its survival during simulated gastric digestion, as determined in this study, it should theoretically be possible to lower the amount that needs to be ingested in cheese by up to a factor

  16. Understanding the fate of sanitation-related nutrients in a shallow sandy aquifer below an urban slum area.

    PubMed

    Nyenje, P M; Havik, J C N; Foppen, J W; Muwanga, A; Kulabako, R

    2014-08-01

    that the cation exchange capacity of the aquifer was low (<6meq/100g) to effectively adsorb NH4. Phosphate transport was, on the other hand, greatly retarded and our results showed that this was due to the adsorption of P to calcite and the co-precipitation of P with calcite and rhodochrosite. Our findings suggest that shallow alluvial sandy aquifers underlying urban slum areas are an important sink of excessive nutrients leaching from on-site sanitation systems.

  17. Evolutionary diversification and characterization of the eubacterial gene family encoding DXR type II, an alternative isoprenoid biosynthetic enzyme

    PubMed Central

    2013-01-01

    Background Isoprenoids constitute a vast family of natural compounds performing diverse and essential functions in all domains of life. In most eubacteria, isoprenoids are synthesized through the methylerythritol 4-phosphate (MEP) pathway. The production of MEP is usually catalyzed by deoxyxylulose 5-phosphate reductoisomerase (DXR-I) but a few organisms use an alternative DXR-like enzyme (DXR-II). Results Searches through 1498 bacterial complete proteomes detected 130 sequences with similarity to DXR-II. Phylogenetic analysis identified three well-resolved clades: the DXR-II family (clustering 53 sequences including eleven experimentally verified as functional enzymes able to produce MEP), and two previously uncharacterized NAD(P)-dependent oxidoreductase families (designated DLO1 and DLO2 for DXR-II-like oxidoreductases 1 and 2). Our analyses identified amino acid changes critical for the acquisition of DXR-II biochemical function through type-I functional divergence, two of them mapping onto key residues for DXR-II activity. DXR-II showed a markedly discontinuous distribution, which was verified at several levels: taxonomic (being predominantly found in Alphaproteobacteria and Firmicutes), metabolic (being mostly found in bacteria with complete functional MEP pathways with or without DXR-I), and phenotypic (as no biological/phenotypic property was found to be preferentially distributed among DXR-II-containing strains, apart from pathogenicity in animals). By performing a thorough comparative sequence analysis of GC content, 3:1 dinucleotide frequencies, codon usage and codon adaptation indexes (CAI) between DXR-II sequences and their corresponding genomes, we examined the role of horizontal gene transfer (HGT), as opposed to an scenario of massive gene loss, in the evolutionary origin and diversification of the DXR-II subfamily in bacteria. Conclusions Our analyses support a single origin of the DXR-II family through functional divergence, in which constitutes

  18. Understanding the fate of sanitation-related nutrients in a shallow sandy aquifer below an urban slum area

    NASA Astrophysics Data System (ADS)

    Nyenje, P. M.; Havik, J. C. N.; Foppen, J. W.; Muwanga, A.; Kulabako, R.

    2014-08-01

    (anammox) given that the cation exchange capacity of the aquifer was low (< 6 meq/100 g) to effectively adsorb NH4. Phosphate transport was, on the other hand, greatly retarded and our results showed that this was due to the adsorption of P to calcite and the co-precipitation of P with calcite and rhodochrosite. Our findings suggest that shallow alluvial sandy aquifers underlying urban slum areas are an important sink of excessive nutrients leaching from on-site sanitation systems.

  19. 1D-myo-inositol 1,4,5-trisphosphate dephosphorylation by rat enterocytes involves an intracellular 5-phosphatase and non-specific phosphatase activity at the cell surface.

    PubMed Central

    Rubiera, C; Velasco, G; Michell, R H; Lazo, P S; Shears, S B

    1988-01-01

    We studied the dephosphorylation of Ins(1,4,5)P3 (inositol 1,4,5-trisphosphate) by permeabilized rat intestinal epithelial cells incubated in a medium resembling intracellular ionic strength and pH. Saponin-permeabilized cells rapidly dephosphorylated Ins(1,4,5)P3 to a mixture of three InsP2 (inositol bisphosphate) isomers, namely Ins(1,4)P2, Ins(1,5)P2 and Ins(4,5)P2. These products were identified by h.p.l.c. analysis after dephosphorylation of both 3H- and 32P-labelled Ins(1,4,5)P3. Ins(1,4)P2 accumulated to about half of the concentration attained by Ins(1,5)P2 and Ins(4,5)P2. Ins(1,4,5)P3 dephosphorylation was inhibited, by up to 75%, by 10 mM-glucose 6-phosphate. In these conditions Ins(1,4)P2 became the predominant product, indicating that glucose 6-phosphate inhibited non-specific dephosphorylation of Ins(1,4,5)P3, at least at the 1- and 4-phosphate groups. Ins(1,4)P2 was further dephosphorylated, and the major InsP (inositol monophosphate) product was Ins4P. Most of the glucose 6-phosphate-inhibitable Ins(1,4,5)P3 phosphatase activity was exposed on the cell surface. The glucose 6-phosphate-insensitive Ins(1,4,5)P3 5-phosphatase activity was not detected until the cells were permeabilized with saponin. This intracellular 5-phosphatase activity was: (i) predominantly associated with the particulate portion of the cell; (ii) strongly inhibited by 10 mM-2,3-bisphosphoglycerate; (iii) insensitive to 50 mM-Li+. Therefore the Ins(1,4,5)P3 5-phosphatase activity in enterocytes appears similar to the 5-phosphatase that has been characterized in a number of cell types. PMID:2848503

  20. ERK5 and Cell Proliferation: Nuclear Localization Is What Matters.

    PubMed

    Gomez, Nestor; Erazo, Tatiana; Lizcano, Jose M

    2016-01-01

    . Although some ERK5 kinase inhibitors have shown antiproliferative activity it is likely that those tumors expressing kinase-inactive nuclear ERK5 will not respond to these inhibitors.

  1. Structure-Function Analysis of the 3' Phosphatase Component of T4 Polynucleotide Kinase/phosphatase

    SciTech Connect

    Zhu,H.; Smith, P.; Wang, L.; Shuman, S.

    2007-01-01

    T4 polynucleotide kinase/phosphatase (Pnkp) exemplifies a family of bifunctional enzymes with 5'-kinase and 3' phosphatase activities that function in nucleic acid repair. T4 Pnkp is a homotetramer of a 301-aa polypeptide, which consists of an N-terminal kinase domain of the P-loop phosphotransferase superfamily and a C-terminal phosphatase domain of the DxD acylphosphatase superfamily. The homotetramer is formed via pairs of phosphatase-phosphatase and kinase-kinase homodimer interfaces. Here we identify four side chains-Asp187, Ser211, Lys258, and Asp277-that are required for 3' phosphatase activity. Alanine mutations at these positions abolished phosphatase activity without affecting kinase function or tetramerization. Conservative substitutions of asparagine or glutamate for Asp187 did not revive the 3' phosphatase, nor did arginine or glutamine substitutions for Lys258. Threonine in lieu of Ser211 and glutamate in lieu of Asp277 restored full activity, whereas asparagine at position 277 had no salutary effect. We report a 3.0 A crystal structure of the Pnkp tetramer, in which a sulfate ion is coordinated between Arg246 and Arg279 in a position that we propose mimics one of the penultimate phosphodiesters (5'NpNpNp-3') of the polynucleotide 3'-PO(4) substrate. The amalgam of mutational and structural data engenders a plausible catalytic mechanism for the phosphatase that includes covalent catalysis (via Asp165), general acid-base catalysis (via Asp167), metal coordination (by Asp165, Asp277 and Asp278), and transition state stabilization (via Lys258, Ser211, backbone amides, and the divalent cation). Other critical side chains play architectural roles (Arg176, Asp187, Arg213, Asp254). To probe the role of oligomerization in phosphatase function, we introduced six double-alanine cluster mutations at the phosphatase-phosphatase domain interface, two of which (R297A-Q295A and E292A-D300A) converted Pnkp from a tetramer to a dimer and ablated phosphatase activity.

  2. ERK5 and Cell Proliferation: Nuclear Localization Is What Matters

    PubMed Central

    Gomez, Nestor; Erazo, Tatiana; Lizcano, Jose M.

    2016-01-01

    . Although some ERK5 kinase inhibitors have shown antiproliferative activity it is likely that those tumors expressing kinase-inactive nuclear ERK5 will not respond to these inhibitors. PMID:27713878

  3. The PIKfyve–ArPIKfyve–Sac3 triad in human breast cancer: Functional link between elevated Sac3 phosphatase and enhanced proliferation of triple negative cell lines

    SciTech Connect

    Ikonomov, Ognian C. Filios, Catherine Sbrissa, Diego Chen, Xuequn Shisheva, Assia

    2013-10-18

    Highlights: •We assess PAS complex proteins and phosphoinositide levels in breast cancer cells. •Sac3 and ArPIKfyve are markedly elevated in triple-negative breast cancer cells. •Sac3 silencing inhibits proliferation in triple-negative breast cancer cell lines. •Phosphoinositide profiles are altered in breast cancer cells. •This is the first evidence linking high Sac3 with breast cancer cell proliferation. -- Abstract: The phosphoinositide 5-kinase PIKfyve and 5-phosphatase Sac3 are scaffolded by ArPIKfyve in the PIKfyve–ArPIKfyve–Sac3 (PAS) regulatory complex to trigger a unique loop of PtdIns3P–PtdIns(3,5)P{sub 2} synthesis and turnover. Whereas the metabolizing enzymes of the other 3-phosphoinositides have already been implicated in breast cancer, the role of the PAS proteins and the PtdIns3P–PtdIns(3,5)P{sub 2} conversion is unknown. To begin elucidating their roles, in this study we monitored the endogenous levels of the PAS complex proteins in cell lines derived from hormone-receptor positive (MCF7 and T47D) or triple-negative breast cancers (TNBC) (BT20, BT549 and MDA-MB-231) as well as in MCF10A cells derived from non-tumorigenic mastectomy. We report profound upregulation of Sac3 and ArPIKfyve in the triple negative vs. hormone-sensitive breast cancer or non-tumorigenic cells, with BT cell lines showing the highest levels. siRNA-mediated knockdown of Sac3, but not that of PIKfyve, significantly inhibited proliferation of BT20 and BT549 cells. In these cells, knockdown of ArPIKfyve had only a minor effect, consistent with a primary role for Sac3 in TNBC cell proliferation. Intriguingly, steady-state levels of PtdIns(3,5)P{sub 2} in BT20 and T47D cells were similar despite the 6-fold difference in Sac3 levels between these cell lines. However, steady-state levels of PtdIns3P and PtdIns5P, both regulated by the PAS complex, were significantly reduced in BT20 vs. T47D or MCF10A cell lines, consistent with elevated Sac3 affecting directly or

  4. Annexin 2 Binding to Phosphatidylinositol 4,5-Bisphosphate on Endocytic Vesicles Is Regulated by the Stress Response Pathway*

    PubMed Central

    Hayes, Matthew J.; Merrifield, Christien J.; Shao, Dongmin; Ayala-Sanmartin, Jesus; Schorey, Crislyn D’Souza; Levine, Tim P.; Proust, Jezabel; Curran, Julie; Bailly, Maryse; Moss, Stephen E.

    2005-01-01

    Annexin 2 is a Ca2+-binding protein that has an essential role in actin-dependent macropinosome motility. We show here that macropinosome rocketing can be induced by hyperosmotic shock, either alone or synergistically when combined with phorbol ester or pervanadate. Rocketing was blocked by inhibitors of phosphatidylinositol-3-kinase(s), p38 mitogen-activated protein (MAP) kinase, and calcium, suggesting the involvement of phosphoinositide signaling. Since various phosphoinositides are enriched on inwardly mobile vesicles, we examined whether or not annexin 2 binds to any of this class of phospholipid. In liposome sedimentation assays, we show that recombinant annexin 2 binds to phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5P2) but not to other poly- and mono-phosphoinositides. The affinity of annexin 2 for PtdIns-4,5P2 (KD ~5 μm) is comparable with those reported for a variety of PtdIns-4,5P2-binding proteins and is enhanced in the presence of Ca2+. Although annexin 1 also bound to PtdIns-4,5P2, annexin 5 did not, indicating that this is not a generic annexin property. To test whether annexin 2 binds to PtdIns-4,5P2 in vivo, we microinjected rat basophilic leukemia cells stably expressing annexin 2-green fluorescent protein (GFP) with fluorescently tagged antibodies to PtdIns-4,5P2. Annexin 2-GFP and anti-PtdIns-4,5P2 IgG co-localize at sites of pinosome formation, and annexin 2-GFP relocalizes to intracellular membranes in Ptk cells microinjected with Arf6Q67L, which has been shown to stimulate PtdIns-4,5P2 synthesis on pinosomes through activation of phosphatidylinositol 5 kinase. These results establish a novel phospholipid-binding specificity for annexin 2 consistent with a role in mediating the interaction between the macropinosome surface and the polymerized actin tail. PMID:14734570